Uses of thioredoxin

ABSTRACT

The present invention relates to the use of thioredoxin as, inter alia, a cell growth stimulator, as well as a screen for agents that are useful in reducing or preventing thioredoxin-associated apoptosis inhibition and agents that are useful in inhibiting thioredoxin stimulated cell growth.

B. CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 10/600,957, filedJun. 20, 2003, which is a continuation of U.S. application Ser. No.06/875,578, filed Jun. 6, 2001, now U.S. Pat. No. 6,689,775, issued Feb.10, 2004, which is a continuation of U.S. Ser. No. 09/319,292, filed onJun. 3, 1999, now abandoned, which is a national phase filing based onInternational Application No. PCT/US 97/22292, filed on Dec. 5, 1997,which claimed the benefit of priority from U.S. Provisional PatentApplication Ser. No. 60/031,995, filed on Dec. 6, 1996. Allaforementioned applications are herein incorporated by reference intheir entirety.

A. STATEMENT REGARDING FEDERAL SPONSORSHIP

This invention was made with support from the U.S. government under agrant from the US. National Institutes of Health, contract numberCA48725. The U.S. government has certain rights in the invention.

C. BACKGROUND OF INVENTION

The present invention generally relates to the use of thioredoxin as,inter alia, a cell growth stimulator, as well as a screen for agentsthat are useful in reducing or eliminating thioredoxin-associatedapoptosis inhibition and agents that are useful in inhibitingthioredoxin stimulated cell growth.

Thioredoxin is a low molecular weight (M_(r) 11,000-12,000) redoxprotein found in both prokaryotic and eukaryotic cells. (Holmgren A., J.Biol. Chem., 264:13963-13966, 1989), that undergoes reversible thiolreduction by the NADPH-dependent enzyme thioredoxin reductase. Humanthioredoxin, which has 5 cysteine (Cys) residues, is a 11.5 kDa proteinwith 27% amino acid identity to E. coli thioredoxin. Human thioredoxincontains 3 additional Cys residues not found in bacterial thioredoxinthat give it unique biological properties. (Gasdaska P Y, et al.,Biochem. Biophys. Acta., 1218:292-296, 1994). Cys32 and Cys35 are theconserved catalytic site cysteine residues that undergo reversibleoxidation to cystine. Cys92, Cys69 and Cys73 are found in mammalian butnot in bacterial thioredoxins, Cys73 appears to be particularlyimportant for maintaining the biological activity of thioredoxin in anoxidizing environment. Thioredoxin reduces a variety of intracellularproteins including enzymes such as ribonucleotide reductase which isimportant for DNA synthesis, and critical Cys residues in transcriptionfactors such as NF-KB, AP-1 and the glucocorticoid receptor, thus,altering their binding to DNA. In addition to its intracellular actions,human thioredoxin has remaskable extracellular cell growth stimulatingproperties. It has been reported (Gasdaska P Y, et al., Biochem.Biophys. Acta., 1218:292-296, 1994) that thioredoxin is identical to agrowth factor reported to be secreted by human HTLV-1 transformedleukemia cell lines (Fox J A, et al., Proc. Natl. Acad. Sci. USA,84:2663-2677, 1987). It has also been found that human recombinantthioredoxin will stimulate the growth of a wide variety of fibroblastand human solid tumor cell lines in culture (Gasdaska J R, et al., CellGrowth Differ., 6:1643-1650, 1995; Oblong J E, et al., J. Biol. Chem.,269:11714-11720, 1994). E. coli thioredoxin does not stimulate cellproliferation.

Thioredoxin was first studied for its ability to act as reducingco-factor for ribonucleotide reductase, the first unique step in DNAsynthesis. (Laurent T C, et al., 15 J. Biol. Chem., 239:3436-3444, 1964)More recently thioredoxin has been shown to exert redox control over anumber of transcription factors modulating their binding to DNA andthus, regulating gene transcription. Transcription factors regulated bythioredoxin include NF-KB (Matthews J R, et al., Nucl. Acids Res.,20:3821-3830, 1992), TFIIIC (Cromlish J A, et al., J, Biol. Chem.,264:18100-18109, 1989), BZLFI (Bannister 20 AJ, et al., Oncogene,6:1243-1250, 1991), the glucocorticoid receptor (Grippo J F, et al., J.Biol. Chem., 258:13658-13664, 1983) and, indirectly through a nuclearredox factor Ref-1/HAPE, thioredoxin can regulate AP-1 (Fos/Junheterodimer) (Abate C, et al., Science 249:1157-1161, 1990). Thioredoxinis also a growth factor with a unique mechanism of action.

Human thioredoxin has been sequenced and cloned. (Gasdaska P Y, et al.,Biochem. Biophys. Acta., 1218:292-296, 1994; Deiss L P, et al., Science252:117-120, 1991). It has been shown that the deduced amino acidsequence of thioredoxin is identical to that of a previously knownprotein called eosinophil cytotoxicity stimulating factor (Silberstein DS, et al. Biol. Chem., 268:9138-9142, 1993) or adult T-cellleukemia-derived factor (ADF) (Gasdaska P Y, et al., Biochem. Biophys.Acta., 1218:292-296, 1994). ADF has been reported to be secreted byvirally transformed leukemic cell lines and to stimulate their growth(Yodoi J, et al., Adv. Cancer Res., 57:381-411, 1991). Theseobservations have been extended to show that human recombinantthioredoxin stimulates the proliferation of both normal fibroblasts anda wide variety of human solid and leukemic cancer cell lines. (GasdaskaJ R, et al., Cell Growth Differ., 6:1643-1650, 1995) Powis G, et al.,Oncol. Res., 6:539-544, 1994; Oblong J E, et al., J. Biol. Chem.,269:11714-11720, 1994). It has been shown that thioredoxin stimulatescell proliferation by increasing the sensitivity of the cells to growthfactors secreted by the cells themselves. (Gasdaska J R., et al., CellGrowth Differ., 6:1643-1650, 1995).

Recombinant modified thioredoxins, otherwise called mutant thioredoxins,have been developed, but no indications of uses were known in the artfor any particular mutant form. In a wild type thioredoxin, the cysteine(Cys) residues at the conserved -Cys32-Gly-Pro-Cys35-Lys active site ofthioredoxin undergo reversible oxidation-reduction catalyzed by theNADPH-dependent flavoprotein thioredoxin reductase, (Luthman M, et al.,Biochem., 21:6628-6633, 1982). It has been reported that mutation of theactive site Cys32 and Cys35 residues to serine (Ser) residues, eithersingly or together (C32S/C35S thioredoxin), results in a compound thatis redox inactive (i.e., it is not a substrate for reduction bythioredoxin reductase) and that does not stimulate cell proliferation(Oblong J E, et al., J. Biol. Chem., 269:11714-11720, 1994).

Thioredoxin mRNA has been found to be over expressed by some human tumorcells (Gasdaska P Y, et al., Biochem. Biophys. Acta., 1218:292-296,1994; Grogan T, et al., Cancer Res., 1997, in press) and since it issecreted from cells by a leaderless secretary pathway (Rubartelli. A, etal., J. Biol. Chem., 267:24161-24164, 1992) it could be a growth factorfor some human cancers (Gasdaska J R, et al., Cell Growth Differ.,6:1643-1650, 1995). However, the mechanism for cell growth stimulationby thioredoxin mRNA has been examined and found not to promote cellgrowth. Recombinant human thioredoxin is not taken up by cells and doesnot bind to high affinity cell surface receptors but appears to enhancethe sensitivity of cells to endogenously produced or other growthfactors, a mechanism termed voitocrine (Greek, voithos=helper) (GasdaskaJ R, et al., Cell Growth Differ., 6:1643-1650, 1995).

The in vitro cell growth stimulating activity of human thioredoxin hasbeen previously reported for human lymphoid and solid tumor cancer cells(Gasdaska J R, et al., Cell Growth Differ., 6:1643-1650, 1995; Oblong JE, et al., J. Biol. Chem., 269:11714-111720, 1994) and for mousefibroblast cells (Oblong J E, et al., J. Biol. Chem., 269:11714-11720,1994). The production of a Cys⁷³->Ser mutant thioredoxin has beenpreviously reported. In one study it did not act like wild-typethioredoxin as a component of a complex cell growth stimulating factorcalled “early pregnancy factor” (Tonissen K, 10 et al., J. Biol. Chem.,268:22485-22489, 1993). In another study it was reported that Cys⁷³->Sermutant thioredoxin did not form a dimer, but cell growth stimulatingactivity by the mutant thioredoxin was not investigated in this study(Ren X, et al., Biochem., 32:9701-9705, 1993). However, the ability ofthe Cys73->Ser mutant and other mutant thioredoxins to stimulate cellproliferation has not been reported. There have been no prior reports ofadministration of mutant thioredoxins in vivo.

It has been known that certain human tumor cells were found toover-express thioredoxin mRNA compared to normal lung tissue from thesame subject (Gasdaska P Y, et al., Biochem. Biophys. Acta.,1218:292-296, 1994). It has also been known that human primarycolorectal tumors have exhibited elevated levels of thioredoxin mRNAcompared to normal colonic mucosa (Berggren M, et al., Anticancer Res.,16:3459-3466, 1996). It has not been known, that thioredoxin protein waspresent in certain human tumor cells, and it has not been known thatthioredoxin protein played any role in preventing or enhancing tumorcell growth.

While thioredoxin itself is known, its use in identifying agents thatinhibit cell growth stimulated by thioredoxin has not been previouslyshown.

Human thioredoxin reductase has been characterized as a protein (OblongJ E, et al., Biochem., 32:7271-7277, 1993). In addition, the generalproperties and the cDNA base sequence of human thioredoxin reductase isknown in the art. However, it has not been disclosed or suggested in theart that thioredoxin reductase be used as an anti-tumor drug target.

The myelodysplastic syndromes (MDS) are a heterogeneous class of lifethreatening diseases characterized by ineffective hematopoiesis andprogressive, refractory cytopenia (List A F, et al. J. Clin. Oncol.,8:1424-1441, 1990). Transformation to acute leukemia may occur inone-third of the patients. The underlying defect is decreasedmultilineage progenitor cell growth associated with decreasedsensitivity to growth factor stimulation (Merchav S, et al., Leukemia,5:340-346, 1991). Very high doses of recombinant granulocyte-macrophagecolony stimulating factor (GM-CSF) and recombinant human granulocytecolony stimulating factor (G-CSF) can ameliorate neutropenia but do notimprove red blood cell or platelet function (List A F, et al., J. Clin.Oncol., 8:1424-1441, 1990). Although IL-3 displays multilineageprogenitor stimulatory effects in normal marrow clinical trials haveshown limited ability to improve hematopoiesis in MDS (List A F, et al.,Blood, 82 (Suppl. 1):377a, 1993). Thus, current treatment for MD islimited by the ability of cytokines to stimulate hematopoieticprogenitor cells and the decreased sensitivity of these cells to growthfactors.

D. SUMMARY OF THE INVENTION

The present invention relates to the use of thioredoxin as, inter alia,a cell growth stimulator, as well as a screen for agents that are usefulin reducing or preventing thioredoxin-associated apoptosis inhibition intumor cells and agents that are useful in inhibiting thioredoxinstimulated growth of tumor cells.

A non-limiting embodiment of the invention involves a method ofinhibiting minor cell growth in a tumor cell that over-expressesthioredoxin comprising contacting said tumor cell with a cell growthinhibiting effective amount of an inhibitor of thioredoxin expression.Such agents can include, inter alia, small molecular compounds thatcomplex with and interfere with the biological action of thioredoxin,preferably those that complex with active Cys residues, antisenseinhibitors of thioredoxin expression, antibodies, or inhibitors ofnucleic acid expression.

A further non-limiting embodiment of the invention involves a method ofreducing inhibition of apoptosis in tumor cells that over-expressthioredoxin comprising contacting said tumor cells with an effectiveamount of an agent that inhibits thioredoxin. Such agents can include,inter alia, antibodies to this redoxin, compounds that inhibit theactivity of this redoxin, preferably those that inhibit the activity ofactive Cys residues in the protein, cross-linking agents and the like.

A further non-limiting embodiment of the invention involves a method ofidentifying an agent that inhibits tumor cell growth in cells thatover-express thioredoxin comprising measuring thioredoxin expression oractivity in a first sample of said cells; contacting a second sample ofsaid cells with an agent to be tested; measuring expression or activityof thioredoxin in said second sample; comparing expression or activityof thioredoxin in said first sample and said second sample; whereby adecrease in expression or activity of thioredoxin in said second sampleis indicative of an agent that inhibits tumor cell growth.

A further non-limiting embodiment of the invention involves a method ofidentifying an agent that reduces inhibition of apoptosis in a tumorcell that over-expresses thioredoxin comprising measuring thioredoxinexpression or activity in a first sample of said cells; contacting asecond sample of said cells with an agent to be tested; measuringexpression or activity of thioredoxin in said second sample; comparingexpression or activity of thioredoxin in said first sample and saidsecond sample; whereby a decrease in expression or activity ofthioredoxin in said second sample is indicative of an agent that reducesinhibition of apoptosis.

A further non-limiting embodiment of the invention involves a method ofidentifying an agent that reduces thioredoxin induced inhibition ofapoptosis in a tumor cell growth.

A further non-limiting embodiment of the invention involves a method ofstimulating cell growth comprising introducing a nucleic acid encoding ahuman thioredoxin having Ser at amino acid reside 73 under conditionswhereby said nucleic acid is expressed.

A further non-limiting embodiment of the invention involves acomposition comprising an agent that is useful in reducing oreliminating thioredoxin-associated apoptosis inhibition and anacceptable carrier.

A further non-limiting embodiment of the invention involves acomposition comprising an agent that is useful in inhibiting thioredoxinstimulated cell growth and an acceptable carrier.

The present invention is based, at least in part, on the discovery thatthioredoxin protein is over-expressed in certain human tumor cells; thatthioredoxin stimulates the growth of cancer cells; that thioredoxininhibits apoptosis; that thioredoxin is over-expressed in some humanprimary tumors and is correlated with increased tumor cell growth anddecreased apoptosis; and that agents that inhibit thioredoxin also haveanti-tumor activity.

The present invention involves the new uses of thioredoxin, thioredoxinreductase, and mutant forms of thioredoxin for use in screening foranti-tumor agents. It has not been known in the art to use thioredoxinand/or thioredoxin reductase in a screening assay for anti-thioredoxinand/or anti-thioredoxin reductase agents for use as anti-tumorcompounds.

The present invention further relates to the use of thioredoxin and/orthioredoxin reductase antibodies for use as anti-tumor agents.

The present invention further relates to the use of anti-sensethioredoxin or anti-sense thioredoxin reductase compounds for use asanti-tumor agents.

The present invention further relates to the use of thioredoxin nucleicacid probes and/or thioredoxin antibodies in a diagnostic assay forcertain cancers.

The present invention further relates to the use of thioredoxin as atarget for agents to be used in combination with existing and newtreatment therapies, such as drugs and radiation, that reduce or preventthe thioredoxin-induced inhibition of apoptosis in tumor cells or toincrease the sensitivity of tumor cells to these modalities.

In addition, mutant forms of thioredoxin provide proteins withadditional growth stimulating activity.

These and still further objects as shall hereinafter appear are readilyfulfilled by the present invention in an unexpected manner as will bereadily discerned from the following detailed description of thepreferred embodiments of the invention, especially when read inconjunction with the accompanying drawings.

In contrast to the present invention, none of the above cited referencesteach or suggest the use of thioredoxin protein according to the claimedinvention.

E. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chart that illustrates the stimulation of human bonemarrow colony formation by Cys73->Ser mutant thioredoxin in accordancewith the present invention, wherein the effects of Cys73->Serthioredoxin on colony formation are shown by (o) multilineageprogenitors (CFU-GEMM); (•) erythroid progenitors (BFU-E): and (∇)myeloid progenitors (CFU-GM), measured over 10 days as described.(Values represent the mean of 4 determinations and bars represent S.D.)

FIG. 2 shows a chart that illustrates potentiation of IL-2 induced MCF-7breast cancer cell growth by Cys⁷³->Ser mutant thioredoxin in accordancewith the present invention, wherein cells were growth arrested for 48hr. in medium with 0.5% serum (10 cells) then stimulated in the absenceof medium with either IL-2 or Cys³²->Ser mutant thioredoxin at theconcentrations shown and cell number was measured after 48 hr. (Eachpoint represents the mean of 3 determinations and bars represent S.E.,and the dotted line shows stimulation by 10% serum.)

FIG. 3 shows a chart that illustrates the inhibition of thioredoxinstimulated MCF-7 cell growth by receptor antibodies in accordance withthe present invention, wherein cell proliferation was measured asdescribed in FIG. 2; the concentrations of agents used were Cys⁷³->Sermutant thioredoxin (Thioredoxin) 1 μM, monoclonal antibodies to FGFreceptor, IL-2-receptor and EGF-receptor 4 μg/ml, and EGF 10 nM; and theEGF and EGFR were added as a negative control. (Values represent themean of 3 determinations and bars represent S.E., and the dotted lineshows the effect of 10% serum alone.)

FIGS. 4A-B illustrate comparative charts showing the effects ofthioredoxin and C32S/C35S cDNA transfection on proliferation of MCF-7cells. In FIG. 4A, 3×10⁴ cells were plated in 3.8 cm² plastic culturedishes in DMEM with 10% fbs and cell number measured 7 days later. InFIG. 413, 10 cells were plated in 2 cm² wells containing soft agaroseand colonies measuring >60 microns counted 7 days later. (Control, theNeol vector alone transfected MCF-7 cells; Thioredoxin 9, Thioredoxin12, and Thioredoxin 20, MCF-7 cells transfected with human ThioredoxincDNA; Serb 4, Serb 15, and Serb 19, MCF-7 cells transfected withC32S/C35S cDNA. Values are the mean of 3 determinations and bars areS.E. **indicates p<0.01 compared to vector-alone transfected cells.)

FIG. 5 illustrates a chart showing the growth of Trx andC32S/C35S-transfected MCF-7 breast cells in Scid mice. Female Scid miceimplanted s.c. 2 days previously with a 21 day release pellet of 0.25 mg17-β estradiol were injected subcutaneously with 2×10⁶ transfected MCF 7cells in 0.1 ml 0.9%, NaCl and 0.1 ml matngel. (O) represents MCFneo,pDC304 vector-alone transfected MCF 7 cells; (▾) represents Trx 12,thioredoxin transfected cells; (▪) represents Trx 20, thioredoxintransfected cells; (∇) represents Serb 4, C32S/C35S transfected cells;and (□) represents Serb 15, C32S/C35S transfected cells. There were 4mice per group. Tumor growth was measured twice a week for 40 days. The17-β-estradiol pellet was replaced at 21 days. Values are mean and bars,SE.

FIG. 6 illustrates an autoradiogram showing Northern analysis of NIH 3T3cells stably transfected with cDNA for human Trx and a redox-inactivemutant human Trx (C32S/C35S) hybridized with a full-length ³²P-labeledhuman Trx cDNA. The bottom band is endogenous mouse Trx mRNA, and thetop band is the transfected human Trx mRNAs. The blots illustratecolumns of NIH 3T3, wild-type NIH 3T3 cells; NeoC, cells transfectedwith the pRXneo vector; ThioAD and Thio6, cells transfected withC32S/C35S Trx in the pDC304neo vector; and Thio 9, cells transfectedwith human Trx cDNA in the pDC304neo vector. Values on right aremolecular weight markers (in kb). Values below are the ratios oftransfected Trx mRNA to endogenous Trx mRNA determined by phosphorimageranalysis.

FIG. 7 illustrates a chart showing the effects of transfection with Trxor C32S/C35S cDNA on the growth of Nit-H 3T3 cells. Cells were plated inplastic dishes at a density of 2×10⁴ cells/cm²—in DMEM with 10% FBS andcell number measured daily. , NeoC vector alone-transfected cells. Theapparent decrease in the number of cells after day 3 is due todetachment of cells from the plastic surface: ∇, ▾, and Δ, Thio6,ThioAD, and Thio9 cells transfected with Trx cDNA; □ and ▪ NIHBH andNIHBF: cells transfected with C32S/C35S cDNA. Values are the mean ofthree determinations: bars, SE. *, P<0.05 compared with vectoralone-transfected cells, shown for days 3 and 4 only. The study istypical of three repeat experiments.

FIG. 8 illustrates a chart showing the stimulation of the proliferationof MCF-7 human breast cancer cells and mouse NIH 3T3 cells by human Trx.Cells were growth arrested in DMEM containing 0.5% FBS for 48 h so thatthere were 0.4×10⁵ cells, at which time the medium was replaced withfresh DMEM with (▪) or without (□) 1 μM human Trx. Cell numbers weremeasured 48 h later. Values are the mean of three determinations: bars,SE.

FIGS. 9A-B illustrate comparative autoradiograms showing Northernanalysis of MCF-7 breast cancer cells stably transfected with cDNA forwild-type Trx (FIG. 9A) and redox-inactive mutant Trx (C32S/C35S)hybridized with a full-length ³²P-labeled human Trx cDNA (FIG. 9B).MCFneo are cells transfected with pDC304 vector alone. The blotsillustrate columns of MC Fneo, Trx20, Trx 14, Trx 12, and Trx9 in FIG.9A and Serb 15, Serb 19, Serb 17, Serb 5, Serb 4 and MCFneo in FIG. 9B.The bottom band is endogenous Trx mRNA, and the top band is thetransfected Trx mRNAs. Values below each column are the ratios oftransfected Trx mRNA to endogenous Trx mRNA determined by phosphorimageranalysis.

FIGS. 10A-D illustrate light microscopy of Trx and C32S/C35ScDNA-transfected MCF-7 breast cancer cells. The cells were grown to 75%confluence on glass coverslips, fixed with methanol, stained with aRomanowski-type dye (Diff-Quick, Baxter), and observed with a×100 oilimmersion objective. In FIG. 1A, pDC304 vector alone-transfected MCF-7cells; FIG. 10B, Trx 20 Trx-transfected MCF-7 cells; FIG. IOC, Serb 4C32S/C35S-transfected MCF-7 cells; and FIG. 10D, Serb 19C32S/C35S-transfected MCF-7 cells.

FIGS. 11A-B illustrate comparative charts showing the effects of Trx andC32S/C35S cDNA transfection on proliferation of MCF-7 cells. In FIG. 1A,cells (3×10) were plated in 3.8m-cm² plastic culture dishes in DMEM with10% FBS, and cell number was measured 7 days later. In FIG. 11B, cells(10⁴) were plated in 2-cm-wells containing soft agarose, and coloniesmeasuring >60 μm were counted 7 days later. In both FIGS. 11A and B, thechart shows control Neol vector alone-transfected MCF-7 cells, Trx 9,Trx 12, and Trx 20, MCF-7 cells transfected with human Trx cDNA: Serb 4,Serb 15, and Serb 19, MCF-7 cells transfected with C32/C35S cDNA. Valuesare the mean of three determinations: bars, SE, **, P<0.01 compared withvector alone-transfected cells.

FIG. 12 illustrates a chart showing the growth of Trx andC32S/C35S-transfected MCF-7 breast cancer cells is Scid mice. FemaleScid mice implanted s.c. 2 days previously with a 21-day release pelletof 0.25 mg of 17-β-estradiol were injected s.c. with 2×10⁶ transfectedMCF-7 cells in 0.1 ml of 0.9% NaCI and 0.1 ml of Matrigel. o, MCFneo,pDC304 vector alone-transfected MCF-7 cells: ▾, Trx 12, Trx-transfectedcells; ▪, Trx 20, thioredoxin-transfected cells: ▾. Serb 4,C32S/C35S-transfected cells; □, Serb 15, C32S/C35S-transfected cells.There were four mice per group. Tumor growth was measured twice a weekfor 40 days. The 17-β-estradiol pellet was replaced at 21 days. Valuesare mean. Bars, SE.

FIG. 13 illustrates a chart showing the stimulation of MCF-7 breastcancer cell growth by fresh and aged Trx and C73S. MCF-7 cells weregrowth arrested and the stimulation of cell proliferation measured over2 days using 1 μM Trx or C73S that was fresh or had been stored as a 25μM stock solution without reducing agent for 5 days or 90 days at 4′.Also shown for reference is the effect of 10% fetal bovine serum. Eachvalue is the mean of 3 determinations, and bars are SEM.

FIG. 14 illustrates a chart showing the reduction of aged Trx bythioredoxin reductase. The incubation mixture contained 0.1 M HEPESbuffer, pH 7.6, 5 mM EDTA, 17 μM insulin, 100 μM NADPH, 15 μg/ml humanthioredoxin reductase. Traces show the oxidation of NADPH at 340 nm atroom temperature with: Line A represents 1 μM fresh Trx; Line Brepresents 30 nM fresh Trx; Line C represents 90-day aged 1 μM Trx; andLine D represents 1 μM. 90-day aged Trx and 30 nM fresh Trx.

FIG. 15 illustrates a chart showing the effect of H₂O₂ on the reductionof Trx (filled bars) and C73S (open bars) by thioredoxin reductase. Trxsolutions were treated with varying amounts of H₂O₂ for 18 hr. at roomtemperature. Reductase activity was measured by adding treated samplesto a solution of 0.1 M HEPES buffer, pH 7.6, 5 mM EDTA, 17 μM. insulin,100 μM NADPH, 15 μg/ml human thioredoxin reductase and measuring therate of NADPH oxidation at 340 nm at room temperature. One hundredpercent of thioredoxin reductase activity is defined as 0.1 absorbanceunit/min/mM Trx or C73S Trx H₂O₂ had no effect on the oxidation ofNADPH.

FIG. 16 illustrates an electrophoretic analysis showing the effect ofstorage on Trx studied by SDS-PAGE. Protein was stained with silverstain. Lane 1 represents fresh Trx; lane 2 represents Trx 48 hr. at roomtemperature without DTT; lane 3 represents Trx 7 days at roomtemperature without DTT; and lane 4 represents Trx stored 90 days at 4′without DTT. Position of molecular mass markers in kDa are shown on theleft.

FIG. 17 illustrates an electrophoretic analysis showing the oxidationand alkylation of Trx studied by SDS-PAGE. Lane 1 represents fresh Trx;lane 2 represents Trx stored at room temperature without DTT for 7 days;lane 3 represents Trx as in lane 2 treated with 3 mM 2-mercaptoethanol;lane 4 represents fresh Trx treated with 1 mM N-ethylmaleimide; lane 5represents fresh Trx treated with 1 mM diamide; lane 6 represents freshTrx treated with 2:1 (v:v) H₂O; lane 7 represents Trx as in lane 5treated with 3 mM 2-mercaptoethanol; and lane 8 represents Trx as inlane 6 treated with 10 mM 2-mercaptoethanol. Position of molecular massmarkers in kDa are shown on the left.

FIG. 18 illustrates an electrophoretic analysis showing the oxidationand reduction of mutant Trxs studied by SDS-PAGE. Lane 1 representsfresh Trx; lane 2 represents fresh C73S Trx; lane 3 represents freshC32S/C35S Trx; lane 4 represents C73S Trx treated with 1 mM diamide;lane 5 represents C73S Trx as in lane 4 treated with 10 mM DTT; lane 6represents C32S/C35S Trx treated with 1 mM diamide; and lane 7represents C32S/C35S Trx as in lane 6 treated with 10 mM DTT. Positionof molecular mass markers in kDa are shown on the left and right.

FIG. 19 illustrates the position of cysteines in human Trx. Ribbons andball-and-stick representation showing the relative positions of Cys³²,Cys³⁵, Cys⁶², Cys⁶⁹ and Cys⁷³, based on the crystal coordinates for thewild type reduced protein (Weichsel A, et al., Structure 4:735-751,1996). None of the thiols are in a position for disulfide bond formationexcept for the redox active pair Cys³² and Cys³⁵. The intermoleculardisulfide bond requiring the least distortion in the protein would bebetween Cys³² and Cys⁷³. The sulfhydryls for these residues are 9.1 Åapart in the model, but could possibly approach each other through localdistortions in nearby residues. Both residues are in loops, makingnecessary distortions of energetically lower cost. The region near Cyshas already been shown to adopt alternate conformations (Weichsel. A, etal., Structure 4:735-751, 1996), in support of this possibility. Thisfigure was made with MOLSCRIPT (Sato N, et al., J. Immunol,154:3194-3203, 1995).

FIG. 20A shows a Northern blot hybridization analysis of total RNAextracted from: wild-type mouse WEH17.2 cells; from pDC304neovector-alone transfected WEH17.2 cells (Neo); and from therrx-transfected WEH17.2 clones Trx5 and Trx6. A full-length ³²P-labeledtrx cDNA probe was used for hybridization, Top band, transfected humanTrx mRNA; bottom band, mouse Trx mRNA. The values on the right show theposition of molecular weight markers (kb).

FIGS. 20B(1)-(4) illustrate fluorescence immunohistochemical staining ofTrx in cells using immunoaffinity-purified rabbit antihuman Trxpolyclonal antibody, biotinylated goat antirabbit IgG; fluoresceinstreptavidin, and laser scanning confocal microscopy. FIG. 20B(1)represents wild-type WEH17.2 cells; FIG. 20B(2) represents pDC304neovector-alone transfected WEH17.2 cells; FIG. 20B(3) represents Trx5 trxtransfected cells; and FIG. 20B(4) represents Trx6 trx-transfectedcells.

FIGS. 20C(1)-(2) illustrate a fluorescence immunohistochemical stainingof Trx using Cy5-streptavidin fluorochrome and Y0Y0-1 to counterstainnuclear DNA showing that Trx is present in the cytoplasm and the nucleusof wild-type WEH17.2 cells (in FIG. 20C(1)) and Trx6 frx-transfectedcells (in FIG. 20C(2)).

FIG. 21 shows comparative charts illustrating the effects of trx andbcl-2 transfection in WEH17.2 cells on dexamethasone-induced apoptosis.

FIG. 21A shows a chart illustrating apoptosis measured by an ELISA forhistone-associated DNA fragments, expressed as relative nucleosomalenrichment. Wild represents wild-type WEH17.2 cells; Neo representspDC304neo vector-alone-transfected WEHI7.2; W.Hbl2 representsbcl-2-transfected WEHI7.2; and Trx5 and Trx6 represent trx-transfectedWEH17.2 cells. The cells were treated with, 0.01% ethanol vehicle (▪) orI μM dexamethasone (□), and apoptosis was measured 24 h later. Columns,mean of four determinations; bars, SE. *, P<0.05 compared to Neocontrol.

FIGS. 21B(1)-(4) illustrate comparative charts of apoptosis measured byflow cytometry showing typical results. Regions R1, R2, and R3 of thescattergrams represent live nonapoptotic, early apoptic, and lateapoptotic cells, respectively. FIG. 21 B(1) shows pDC304neovector-alone-transfected control cells; FIG. 21B(2) shows pDC304neovector-alone-transfected cells treated 48 h with 1 μM dexamethasone;FIG. 21B(3) shows Trx6 trx-transfected WEH17.2 cells; and FIG. 21 B(4)shows Trx6 cells treated for 48 hr. with 1 μM dexamethasone.

FIG. 22A shows a chart illustrating tumor formation in Scid mice bywild-type WEH17.2 cells (O and); bcl-2-transfected WEH17.2 (W. Hbl2)cells (∇ and ▾); and Trx6 rrx-transfected WEH17.2 cells (□ and ▪).Twenty mice were injected s.c. with 2×10 cells and 0.1 ml matrigel inthe flank. Tumor size was measured every 3-4 days with calipers, andtumor volumes were calculated. Nine days after tumor cell injection,half of the mice were injected s.c. into the opposite flank with 1mg/kg/day dexamethasone (o, ∇, and □) or with vehicle alone (•, ∇, and▪). Mice were euthanized at the first measurement at which tumor volumeexceeded 2 cm³ Data points, mean of mice in each group; bars, SE.Results similar to the Trx6 cells were obtained with Trx5 cells.

FIG. 22B illustrates 1-μm sections of epoxy-embedded tumor samples takenat day 14 stained with toluidine blue O examined by bright-fieldmicroscopy. Spontaneous apoptosis in wild-type WEH1 7.2 tumor are shownin FIG. 22B(1) and Trx6 tumor showing less spontaneous apoptosis areshown in FIG. 22B(2).

FIG. 23 illustrates thioredoxin positive gastric carcinoma (Case 2).FIGS. 23A-B show that hematoxylin and eosin stains (FIG. 23A and FIG.23B) reveal a pleomorphic carcinoma invading the gastric wall. FIGS.23C-D show that the thioredoxin expression (FIG. 23C and FIG. 23D) ispresent in both the nuclei and cytoplasm of tumor cells in malignantglands and in rare associated leucocytes. Thioredoxin expression isabsent in the adjacent stroma (1000× to 400×).

FIG. 24 illustrates thioredoxin negative gastric carcinoma (Case 8).FIG. 24A represents gastric carcinoma with complex glands in lower fieldof view with overlying normal gastric mucosa and submucosa. Hematoxylinand eosin (100×). FIG. 24B represents the same section as shown in FIG.24A, negative control stained after biotin-avidin block to eliminatebiotin receptor affect and stained with substituted irrelevant isotypematched monoclonal antibody (100×, Diaminobenzidine). FIG. 24Crepresents the same section as shown in FIGS. 24A and B showing faintreactivity in normal upper mucosa (+), moderate reactivity in thesubmucosa (++) and the underlying gastric carcinoma appears negative forthioredoxin (0) (100×). FIG. 24D represents a higher magnification ofFIG. 24(C showing detail with gastric pit cells having both nuclear andcytoplasmic stain and absent tumor staining (250×).

FIG. 25 illustrates normal gastric mucosa-thioredoxin and Ki67(proliferation antigen) expression. FIG. 25A shows normal gastric mucosaand gastric pits with underlying muscularis propria hematoxylin andeosin (100×). FIG. 25B shows the same section as shown in FIG. 25A,stained for thioredoxin with faint (+) mucosal staining and moderate(++) gastric pit staining (100×). FIG. 25C shows the same section asshown in FIG. 25B at higher magnification. Note the faint mucosalstaining is solely cytoplasmic, while lower lying gastric pit cells areboth cytoplasmic and nuclear (250×). FIG. 25D shows the nuclear Ki67expression notable in lower mucosa and upper gastric pits (25O×).

FIG. 26 illustrates thioredoxin intense gastric carcinoma related tostrong proliferation and weak apoptosis (Case 4). FIG. 26A shows complexadenocarcinoma cell glands (400×, hematoxylin and eosin). FIG. 26B showsintense thioredoxin expression in gastric carcinoma cells (400×), FIG.26(C shows a high percentage of Ki67 positive cells indicating highproliferation (400×). FIG. 26D shows a rare Td+− apoptotic cell 25indicating weak apoptosis (Tunel assay, 400×, same slide).

FIG. 27 illustrates thioredoxin negative gastric carcinoma related toweak proliferation and strong apoptosis (Case 6). FIG. 27A shows complexadenocarcinoma glands (400×, hematoxylin and eosin). FIG. 27B shows theabsent thioredoxin expression (400×). FIG. 27C shows a low percentage ofKi67 positive cells indicating a low proliferative rate (400×). FIG. 27Dshows a very high Tdt+ apoptotic cell rate indicating strong apoptosis(TUNEL assay, 400×, same slide).

F. DETAILED DESCRIPTION OF THE INVENTION

All of the various publications cited in the present specification areincorporated by reference in their entireties.

1. DEFINITIONS

In order that the invention herein described may be fully understood,the following definitions are provided:

“Nucleotide” means a monomeric unit of DNA or RNA consisting of a sugarmoiety (pentose), a phosphate, and a nitrogenous heterocyclic base. Thebase is linked to the sugar moiety via the glycosidic carbon (1′ carbonof the pentose) and that combination of base and sugar is called a“nucleoside”. The base characterizes the nucleotide. The four DNA basesare adenine (“A”), guanine (“G”), cytosine (“C”), and thymine (“T”). InRNA uracil (“U”) substitutes for T. In double stranded molecules, an Aon one strand pairs with T(U) on the other, and G with C. As isconventional for convenience in the structural representation of a DNAnucleotide sequence only one strand is shown in which A on one strandconnotes T on its complement and G connotes C. DNA comprises deoxyriboseas the sugar while RNA comprises ribose.

“Amino acids” are shown either by a three letter or one letterabbreviation as follows:

Abbreviated Designations Amino Acid A Ala Alanine C Cys Cysteine D AspAspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine HHis Histidine I Ile Isoleocine K Lys Lysine L Leu Leucine M MetMethionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg ArginineS Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y TyrTyrosine

“DNA Sequence” means a linear array of nucleotides connected one to theother by phosphodiester bonds between the 3′ and 5′ carbons of adjacentpentoses.

“Codon” means a DNA sequence of three nucleotides (a triplet) whichencodes through mRNA an amino acid, a translation start signal or atranslation termination signal. The code however, is degenerate, withsome amino acids being encoded by more than one triplet codon. Forexample, the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG allencode the amino acid leucine (“Leu”), TAG, TAA and TGA are translationstop signals and ATG is a translation start signal.

“Proteins”, “peptides” and “poly peptides” are composed of a lineararray of amino acids connected one to the other by peptide bonds betweenthe alpha-amino and carboxyl groups of adjacent amino acids.

“Genome” means the entire DNA of an organism, cell or a virus. Itincludes, inter alia, the structural genes coding for polypeptides, aswell as regulatory regions including operator, promoter, terminator,enhancer and ribosome binding and interaction sequences.

“Gene” means a DNA sequence which encodes through its template ormessenger RNA (“mRNA”) a sequence of amino acids characteristic of aspecific polypeptide.

“cDNA” means a complementary or copy DNA prepared by using mRNA as atemplate for synthesizing the first strand of DNA using reversetranscriptase, an appropriate oligonucleotide primer and a mixture ofnucleotides.

“PCR” means a polymerase chain reaction whereby a specific DNA sequence,either genomic or cDNA, can be preferentially amplified by the enzymeTaq polymerase using synthetic, oligonucleotide sense and antisenseprimers, (which specify a target sequence), a mixture of nucleotides anda temperature thermocycling regime which allows sequential denaturing,annealing and synthesis of the target DNA between the primers.

“Transcription” means the process of producing mRNA from a gene or DNAsequence.

“Translation” means the process of producing a polypeptide from mRNA.

“Expression” means the process undergone by a gene or DNA sequence toproduce a polypeptide and comprises a combination of transcription andtranslation.

“Plasmid” or “phagemid” means a nonchromosomal double-stranded DNAsequence comprising an intact “replicon” such that the plasmid isreplicated in a host cell. When the plasmid is placed within aunicellular organism, the characteristics of that organism may bechanged or transformed as a result of the DNA of the plasmid. Forexample, a plasmid carrying the gene for ampicillin resistance (AMP^(R))transforms a cell previously sensitive to ampicillin into one which isresistant to it. A cell transformed by a plasmid is called a“transformant”.

“Recombinant DNA Molecule” or “Hybrid DNA” means a molecule consistingof segments of DNA from different genomes which have been joinedend-to-end outside of living cells and able to be maintained in livingcells.

“Apoptosis” is programmed cell death activated by a genetic program toimplement a series of events that cause the death and disposal of acell. This is in contrast to cell death occurring by necrosis, usuallyas a result of injury to the cell.

“Oncogene” is a gene that encodes a protein able to transform cells inculture to induce cancer in animals.

“Over-expression” in the context of determining over-expression ofthioredoxin or recombinant modified thioredoxin is characterized by atwo fold increase or more of the levels of thioredoxin or recombinantmodified thioredoxin in a target sample compared with a control sample.

The following abbreviations may be used throughout the disclosure:

“C32C35S”=Cys³²->Ser³²/Cys³⁵->Ser³⁵

“DTT”=dithiothreitol

“FBS”=Fetal bovine serum

“NEM”=N-ethylmaleimide

“Scid”=Severe combined immunodeficient

“Trx”=thioredoxin

2. USE OF THIOREDOXIN AS AN ONCOGENE

In a non-limiting embodiment of the present invention, NIH-3T3 cellstransfected with human thioredoxin DNA that has been directed to thenucleus of the cells by a nuclear localization signal causes malignanttransformation of the cells.

In a further non-limiting embodiment, stable transfection of mouseWEHI7.2 lymphoid cells with human thioredoxin DNA has been shown toinhibit apoptosis induced by a variety of agents includingglucocorticoid, N-acetylsphingosine, staurosporine, thapsigargin andetoposide, which is similar to the pattern of inhibition of apoptosiscaused by the anti-apoptotic oncogene bcl-2 in these cells. Thethioredoxin transfected WEH17.2 cells form tumors in Scid mice that growmore rapidly and show less spontaneous apoptosis than vector-alone orbcl-2 transfected cells, and are resistant to growth inhibition byglucocorticoid. Therefore, the thioredoxin gene acts as an oncogeneaccording to the standard definition of an oncogene: a gene that encodesa protein able to transform cells in culture or to induce cancer inanimals (Lodish H, et al., “Cancer.” In: Lodish H, Baltimore D, Berk A,Zipursky S L, Matsudaira P, Darnell J, eds. Molecular Cell Biology. NewYork: Scientific American Books, pp. 1258, 1995).

In a further non-limiting embodiment, the thioredoxin gene offers anincreased survival advantage as well as a growth advantage to tumors invivo, unlike the known anti-apoptosis oncogene bcl-2 which offers only asurvival advantage and requires other genetic changes for tumor growth(McDonnell T J, et al., Nature 349:254-256, 1991).

3. THIOREDOXIN IS OVER-EXPRESSED IN CERTAIN HUMAN TUMOR CELLS

It has been discovered that thioredoxin DNA is over-expressed in certainhuman tumor cells resulting in the over production of thioredoxin.According to a technique for reproducibly retrieving antigens forimmunohistochemical studies from archived paraffin, human tissuepathology samples were embedded and used it to study thioredoxin proteinlevels with a panel of human primary gastric carcinoma tissue samples,it has been found that thioredoxin is present in dividing normal basalcrypt cells. It has been further learned that, as the cellsdifferentiate and move down the villi to eventually be shed into thegastric lumen, thioredoxin levels decrease. By stably transfectingmurine NIH 3T3 fibroblast-like cells and human MCF-7 breast cancer cellswith cDNA for human wild-type thioredoxin or with cDNA for aredox-inactive mutant thioredoxin, it has been found that transfectionwith thioredoxin increases the density to which the NIH 3T3 cells growin culture and stimulates anchorage-independent colony formation byMCF-7 breast cancer cells. The redox-inactive mutant thioredoxin actedin a dominant-negative manner, so that transfected MCF-7 cells showedinhibited growth and a reversal of the transformed phenotype, assessedby growth in vitro and in vivo.

It has been shown that stable transfection of mouse NIH 3T3 normalembryonic cells with human thioredoxin cDNA increases their growth rateand cell saturation density in culture (normal NIH 3T3 cells are highlycontact inhibited) which is in vitro evidence of transformation. It hasalso been shown that transfection of MCF-7 human breast cancer and HT-29human colon carcinoma cells with human thioredoxin cDNA increases theircolony formation in soft agarose and tumor growth by HT-29 colon cancercells when the cells are inoculated into immunodeficient (Scid) mice.

Trx was originally studied for its ability to act as a cofactor forribonucleotide reductase, the first unique step in DNA synthesis(Laurent, T. C, et al., J. Biol. Chem., 239:3436-3444, 1964). Human Trxwas subsequently shown to modulate the DNA binding of severaltranscription factors that regulate cell proliferation, includingnuclear factor KB (Hayashi., T, et al., J. Biol. Chem. 268:11380-11388,1993), the glucocorticoid receptor (Grippo, J. F, et al., J. Biol. Chem.258:13658-13664, 1983), and, indirectly through the nuclear redoxprotein Ref-1, activator protein-1 (Fos/Jun heterodimer; Abate, C, etal., Science (Washington D.C.), 249:1157-1161, 1990): Cloning andsequencing of human Trx have shown that it has a predicted amino acidsequence (Gasdaska, P. Y. et al., Biochem. Biophys. Acta., 1218:292-296.1994: Deiss. L. P, et al., Science (Washington D.C.), 252:117-120, 1991)identical to that of a growth factor secreted by virus-transformedleukemic cell lines, termed adult T-cell leukemia-derived factor(Tagaya, Y, et al., J. Immunol, 140:2614-2620, 1988; Wakasugi, N, etal., Proc. Natl. Acad. Sci. USA, 87:8282-8286, 1990). Human Trx, but notbacterial Trx, added to the culture medium stimulates the growth of avariety of normal and cancer cell lines (Wakasugi, N. et al., Proc.Nail. Acad. Sci. USA. 87:8282-8286, 1990; Yodoi, J, et al., Adv. CancerRes., 57:381-411, 1991; Oblong, J. E, et al., J. Biol. Chem.,269:11714-11720, 1994). The added Trx is not taken up by cells(Gasdaska, J R., et al., Cell Growth & Differ., 6:1643-1650, 1995) andappears to stimulate cell growth by enhancing the action of other growthfactors (Gasdaska, J. R., et al., Cell Growth & Differ., 6:1643-1650,1995; Tagaya, Y, et al., EMBO J., 8:757-764, 1989). The redox activityof Trx is required for growth stimulation, and redox-inactive mutantTrxs do not stimulate cell growth (Oblong, J. E, et al., J. Biol. Chem.,269:11714-11720, 1994).

Trx mRNA levels are increased compared with corresponding normal tissuein almost half human primary lung (Gasdaska, P. Y, et al., Biochem.Biophys. Ada., 1218:292-296, 1994) and colon tumors examined (Berggren,M, et al., Anticancer Res., (in press) 1996). Trx protein has beenreported to be increased in human cervical neoplastic squamousepithelial cells (Fujii, S, et al., Cancer (Phila.), 68:1583-1591, 1991)and hepato-cellular carcinoma (Nakamura, H, et al., Cancer (Phila.),69:2091-2097, 1992). Trx is excreted from cells (Ericson, M. L, et al.,Lymphokine Cytokine Res., 11:201-207, 1992; Rubartelli, A, et al., J.Biol. Chem., 267:24161-24164, 1992; Rubartelli, A. et al., Cancer Res.,55:675-680, 1995) using a leaderless secretory pathway. (Rubartelli, A,et al., J. Biol. Chem., 267:24161-24164, 1992), and we have suggestedthat Trx might be a growth factor for some human cancers (Gasdaska. J.R, et al., Cell Growth & Differ., 6:1643-1650, 1995). However, itremains to be unequivocally demonstrated that endogenously produced Trxcan affect cell proliferation. The role Trx plays in the transformedphenotype or cancer cells also is not known.

To provide some answers to these questions, we have stably transfectedmurine NIH 3T3 fibroblast-like cells and human MCF-7 breast cancer cellswith cDNA for human wild-type Trx or with cDNA for a redox-inactivemutant Trx. We have found that transfection with Trx increases thedensity to which the NIH 3T3 cells grow in culture and stimulatesanchorage-independent colony formation by MCF-7 breast cancer cells. Theredox-inactive mutant Trx acted in a dominant-negative manner, so thattransfected MCF-7 cells showed inhibited growth and a reversal of thetransformed phenotype, assessed by growth in vitro and in vivo.

a. Materials and Methods

Human wild-type Trx cDNA and cDNA for C32/C35S redox-inactive Trx, inwhich both active-site cysteine residues are replaced by serine (Oblong,J. E, et al, J. Bio. Chem. 269:11714-11720, 1994), were prepared asdescribed previously (Gasdaska, P. Y. et al., Biochem. Biophys. Ada.,1218:292-296, 1994: Oblong, J. E, et al., J. Biol. Chem.,269:11714-11720, 1994). The cDNAs were cloned into either the KpnI orSacI sites of the pRXneo mammalian transfection vector, underconstitutive control of the Rous sarcoma virus promotor (Dieken, E. S,et al., Mol. Cell. Biol, 12:589-597, 1992), or into the NotI site of thepDC304neo mammalian transfection vector, in which constitutiveexpression is driven by the cytomegalovirus and SV-10 promoters (Gordon,D. A, et al., Exp. Cell Res., 217:368-377, 1995). Correct orientation ofthe cDNAs in the vectors was confirmed by restriction digestion. ThepRXneo and pDC304neo vectors were obtained from Dr. Roger Miesfeld(University of Arizona, Tucson, Ariz.).

Human MCF-7 breast cancer cells and murine NIH 3T3 cells were obtainedfrom the American Tissue Type Collection. (Rockville, Md.), maintainedin DMEM containing 10% FBS under 6% CO₂ at 37° C., and passaged beforeconfluence. NIH 3T3 cells were transfected with Trx:pRXneo,Trx:pDC304neo, C32S/C35S:pDC304neo, or pRXneo alone. MCF-7 cells weretransfected with Trx:pDC304neo, C32S/C35S:pDC304neo, or pDC304neo alone.Transfection used liposomes ofN-[1,2,3-dioleolylpropyl]-N,N,N-trimethylammonium-methylsulfate(Boehringer Mannheim, Indianapolis, Ind.) according to themanufacturer's instructions. Cells were selected by growing for 4 weeksin DMEM with 10% FBS and 400 μg/ml G418 sulfate (Life Technologies,Gaithersburg, Md.). Cell colonies were isolated by trypsinization ontosmall squares of sterile filter paper and expanded by growing in thesame medium. All studies were conducted on clonal cell lines betweenpassages 3 and 10.

Northern hybridization analysis of Trx and C32S/C35S mRNA used afull-length [α-³²P]dCTP-labeled human Trx cDNA probe as describedpreviously (Gasdaska, P. Y, et al., Biochem. Biophys. Ada.,1218:292-296, 1994), and the blots were quantified using aPhosphorImager (Molecular Dynamics, Sunnyvale, Calif.). Transfected Trxand C32S/C35S mRNA levels were expressed relative to endogenous Trx mRNAlevels in the cells. Western analysis of lysates from Trx andC32S/C35S-transfected MCF-7 cells, prepared by sonicating the cells in20 mm Tris-HCl buffer (pH 8.0), 137 mm NaCl, 1 mm MgCl₂, 1 mm CaCl₂, 10%glycerol, 1% Triton X-100, and 1 mm phenylmethylsulfonylfluoride, or inDMEM in which the cells had been incubated at 37° C. for 6 h, wasperformed using immunoaffinity-purified rabbit polyclonal IgG raisedagainst human Trx (Gasdaska, J. R., et al., Cell Growth & Differ.,6:1643-1650, 1995) which also recognizes C32S/C35S. Detection used theenhanced chemiluminescence system (Amersham Life Sciences, Rockford,Ill.), and quantification of autoradiograms was by densitometry (EagleEye II, Stratagene, La Jolla, Calif.).

Colony formation by MCF-7 cells was measured in soft agarose with DMEMand 10% FBS over 7 days, as described previously, (Alley, M. C, et al.,Br. J. Cancer, 52:205-224, 1985).

Growth of cells on plastic surfaces with DMEM and 10% FBS was measureddaily for NIH-3T3 cells over 4 days and for MCF-7 cells over 7 days, asdescribed previously (Powis, G, et al., Biochem. Pharmacol.36:2473-2479, 1987). All cell growth studies were conducted in theabsence of G418 sulfate.

Tumor formation by transfected NIH 3T3 cells was studied by the s.c.injection of 10⁷ transfected cells in 0.1 ml of sterile 0.9% NaCl intothe backs of groups of four male Scid mice or six nude mice. Tumorformation by MCF-7 cells was studied by injecting 2×10⁷ cells in 0.1 mlof sterile 0.9% NaCl and 0.1 ml of Matrigel (Becton Dickinson, Bedford,Mass.) s.c. into the backs of groups of four female Scid mice that hadbeen implanted s.c. 2 days previously with 21-day release pellets of0.25 mg of 17-P-estradiol (Innovative Research, Sarasota, Fla.). The17-P-estradiol pellet was replaced at 21 days. Tumor volume was measuredwith calipers (Geran, R. I, et al., Cancer Chemother. Rep., 3:1-103,1972) twice a week for 40 days. At the end of the study, the animalswere killed, and tumors and other organs were taken for histologicalanalysis.

Statistical analysis was by nonpaired test unless otherwise stated.Tumor growth rates in Scid mice were linearized using the cube root ofthe tumor volume by day for each mouse, and ANOVA was performed usingDunnett's test to determine significant differences form the vectoralone-transfected (control) cell line.

b. Trx and C32S/C35S Transfection of NIH 3T3 Cells

Transfection of mouse NIH 3T3 cells with Trx:pRXneo yielded 6 clonesstably expressing Trx mRNA, transfection with Trx:pDC304 yielded 4clones, and transfection with C32S/C35S:pDC304 neo yielded 12 clones.The levels of transfected mRNA in some of the clones is shown in FIG. 6.The human Trx and C32S/C35S mRNAs were larger than the endogenous mouseTrx mRNA, probably because the transfected Trx mRNAs also containportions of the vector promoter region, the 5′ leader sequence, or thepolyadenylate tail. The level of transfected Trx mRNA expression wasrelatively low, being only 0.2-1.4-fold the endogenous mouse Trx mRNA.Western blotting showed no significant increase in the level of Trxprotein in the cells compared with wild-type or vector alone-transfectedcells (results not shown).

The Trx-transfected NIH 3T3 cells grew at the same rate on a plasticsurface, but reached saturation densities up to twice that of the vectoralone-transfected NIH 3T3 cells (FIG. 7). The vector alone-transfectedcells had the same growth characteristics of wild-type NIH 3T3 cells.NIH 3T3 cells transfected with the redox-inactive C32S/C35S Trx grewmore slowly and reached a lower saturation density on a plastic surfacethan the vector alone-transfected cells. Neither the vectoralone-transfected NIH 3T3 cells nor the Trx or C32S/C35S Trx transfectedcells formed colonies in soft agarose (results not shown).

The ability of transfected NIH 3T3 cells to form tumors when inoculatedinto immunodeficient mice is used to identify neoplastic transforminggenes (Pitot, H. C. Fundamentals of Oncology, p. 149. New York: MarcelDekker, Inc., 1981). When the Trx-transfected NIH 3T3 cells Thio6 orThioAD were injected i.m. into Scid or nude mice, there was no tumorformation over 40 days (results not shown). Thus. Trx expression, atleast at the level obtained in this study, was not, by itself,transforming.

c. Trx and C32/C35S Transfection of MCF-7 Breast Cancer Cells

Human solid cancer cells generally show a greater proliferation responseto added Trx than do mouse fibroblasts (Oblong, J. E, et al., 7. Biol.Chem., 269:11714-11720, 1994; Gasdaska, J. R, et al., Cell Growth &Differ., 6:1643-1650, 1995). This is shown for MCF-7 human breast cancercells compared with NIH 3T3 cells in FIG. 8. Thus, we also studied theeffects of Trx transfection using MCF-7 breast cancer cells.Transfection of MCF-7 cells with Trx:pDC304neo yielded 31 clones thatstably overexpressed Trx mRNA, and transfection with C32S/C35S:pDC304neoyielded 45 clones stably expressing C32S/C35S mRNA. Expression oftransfected mRNAs by some of the clones is shown in FIG. 9. As seenpreviously with the mouse cells, the transfected human Trx mRNAs inMCF-7 cells were larger than endogenous human Trx mRNA. The level of TrxmRNA expression was up to 0.8-fold and C32S/C35S mRNA up to 2.1-fold ofthe endogenous Trx mRNA levels. Light microscopy showed no difference inthe appearance of vector alone-transfected and Trx-transfected MC F-7cells growing on glass coverslips (FIG. 10), and both were similar towild-type MCF-7 cells. In contrast, C32S/C35S-transfected MCF-7 cellsappeared more rounded and had a reduced cytoplasm-to-nucleus ratio.

Quantitative Western immunoblotting showed no significant increase inthe level of Trx protein in the transfected MCF-7 cells compared withvector alone-transfected MCF-7 cells except for one clone (Table 1).There was, however, a significant 60% increase in the secretion of Trxinto the medium by three of the clones compared with the vectoralone-transfected MCF-7 cells. The three other clones showed a 20-50%increase of Trx in the medium, but this was not statisticallysignificant. Thus, it appears that most of the extra Trx and C2S/C35Sproduced by the transfected cells is secreted into the medium.

All of the transfected MCF-7 cells showed linear growth characteristicson plastic surfaces over 7 days. The Trx-transfected cells grew at thesame rate as the vector alone-transfected MCF-7 cells (FIG. 11).However, when grown in the absence of, or with 0.5% FBS for 2 days, theTrx-transfected cell grew at twice the rate of the vectoralone-transfected cells (results not shown). The C32S/C35S-transfectedcells grew at a significantly slower rate that was 56-78% of the vectoralone-transfected cells (FIG. 11). Colony formation was significantlyincreased between 3- and 4-fold for the Trx-transfected MCF-7 cellscompared with the vector alone-transfected cells, and significantlydecreased up to 73% for the C32S/C35S-transfected cells when the cellswere grown in soft agarose. The vector alone-transfected cells showedgrowth characteristics identical to those of wild-type MCF-7 cells underall conditions (results not shown).

d. Tumor Formation by Trx- and C32s/C35s Transfected MCF-7 Cells

The vector alone-transfected MCF-7 cells injected into Scid mice formedtumors that grew at the same rate as nontransfected MCF-7 cells we haveseen in other studies. Trx-transfected MCF-7 cells formed tumors in Scidmice, although they grew at a significantly slower rate than tumorsformed by vector alone-transfected cells: 57% for Trx 12 cells and 38%for Trx 20 cells (both P<0.05 by least squares regression analysis; FIG.12). Tumor formation by the C32S/C35S-transfected MCF-7 cells was almostcompletely suppressed. Tissues from the injection site and other organswere taken for histological examination at the end of the study. Theanimals injected with vector alone or Trx-transfected cells showed largesolid tumors. The animals injected with C32S/C35S-transfected cellsshowed small microscopic tumor cell deposits. There was no evidence oftumor metastasis to other organs in any of the animals. Northernanalysis of the tumor taken from animals injected with Trx-transfectedcells showed the presence of transfected Trx mRNA as determined by itslarge size (results not shown).

TABLE 1 Trx Levels in Transfected Mcf 7 Breast Cancer Cells and MediaClone Cell^(a) Medium MCF-7 vector 1.0 ± 0.0 1.0 ± 0.0 Trx 9 0.9 ± 0.0 1.6 ± 0.5* Trx 12  1.2 ± 0.1* 1.2 ± 0.2 Trx 20 1.1 ± 0.1 1.5 ± 0.2 Serb4 0.9 ± 0.1  1.6 ± 0.6* Serb 15 0.8 ± 0.2 1.3 ± 0.2 Serb 19  1.2 ± 0.1*I.6± Trx or redox-inactive C32S/C35S (Serb)-transfected MCF-7 breastcancer cells (10⁶) were incubated in 5 ml of DMEM for 6 hr. and Trx inthe original cell pellet or in the medium measured by quantitativeWestern immunoblotting. Values are a mean of three separatedeterminations expressed relative to vector alone-transfected cells.^(a)*P < 0.05 by a nonpaired/test compared with vector alone-transfectedcells.

e. Discussion

Trx regulates the redox state and activity of a number of intracellularproteins that control cell growth, including ribonucleotide reductase(Laurent, T. C, et al., J. Biol. Chem., 239:3436-3444, 1964) and the DNAbinding of several transcription factors (Hayashi, T, et al., J. Biol.Chem. 268:11380-11388, 1993; Grippo, J. F, et al. J. Biol. Chem.,258:13658-13664, 1983; Abate, C, et al., Science (Washington D.C.),249:1157-1161, 1990). Recombinant human Trx added to normal and cancercells in culture stimulates their proliferation (Gasdaska, J. R, et al.,Cell Growth & Differ., 6:1643-1650, 1995). However, it has not beendemonstrated that endogenously produced Trx can stimulate cellproliferation. Furthermore, the role Trx may play in malignanttransformation of cells is not known. The present study was undertakento address some of these questions. NIH 3T3 cells transfected with Trxshowed an increased cell saturation density when grown as a monolayer onplastic surfaces. Loss of contact inhibition is a feature of transformedcells (Pitot. H. C., Fundamentals of Oncology, p. 149, New York: MarcelDekker, Inc., 1981), and the cell saturation density of theTrx-transfected NIH 3T3 cells was similar to that seen with othertransformed, weakly tumorigenic mouse 3T3 cell lines (Schlager, J. J, etal., Cancer Res., 53:1338-1342, 1993). However, the Trx-transfected NIH3T3 cells did not form tumors when inoculated into immunodeficient mice.The Trx-transfected MCF-7 cells did not show increased growth on plasticsurfaces in normal FBS, but exhibited significantly increasedanchorage-independent growth measured by colony formation in softagarose. It is surprising that when the Trx-transfected MCF-7 cells weregrown as xenografts in Scid mice, they exhibited decreased growth ratecompared with vector alone-transfected MCF-7 cells. This may be becausehuman Trx can stimulate the immune system of mice so that Trx secretedby the transfected MCF-7 cells might promote some immune rejection, evenby the Scid mice, which, although deficient in mature B and Tlymphocytes, have natural killer-, myeloid-, and antigen-presentingcells (Shultz, L. D. Am. J. Anat., 191:303-311, 1991).

Both NIH 3T3 and MCF-7 breast cancer cells transfected with theC32S/C35S Trx showed slowed growth rates on a plastic surface. Inaddition, colony formation by MCF-7 breast cancer cells in soft agarosewas considerably decreased. When injected into Scid mice, theC32S/C35S-transfected MCF-7 cells formed only microscopic tumors.C32S/C35S is a redox-inactive mutant Trx that acts as competitiveinhibitor of Trx reductase (Oblong, J. E, et al., J. Biol. Chem.,269:11714-11720, 1994). Our X-ray crystallographic studies haveidentified a highly conserved 12 amino acid hydrophobic surface onmammalian, but not bacterial, Trxs, which stabilizes the Cys⁷³-mediateddisulfide-bonded dimer (Weichsel, A, et al., Structure, 4:735-751,1996). The physiological function of this Cys⁷³-Cys⁷³ linked Trx dimeris not known. The surface structure of C32S/C35S is very similar to thatof Trx (Weichsel. A, et al., Structure, 4:735-751, 1996) so thatC32S/C35S is likely to participate in the formation of a heterodimerwith Trx and thus might lower Trx monomer concentrations or affect thebiological activity of the dimer. Unlike wild-type Trx, C32S/C35S doesnot stimulate cell growth when added to the culture medium. C32S/C35Smight also act as a competitive inhibitor to the normal redox-activesubstrates of Trx. Whatever the mechanism, it appears that C32S/C35Sacts in a dominant-negative manner to inhibit the effects of endogenousTrx and, in so doing, inhibits cell growth and reverses the transformedphenotype of MCF-7 breast cancer cells.

Most of the added Trx or C32S/C35S protein, that is produced by thetransfected cells appears to be secreted into the medium. Whether thetransfected Trx is produced in a different compartment to endogenousTrx, allowing it to be secreted, or whether a constant proportion of Trxis secreted is not known. Trx is known to be secreted from cells by aleaderless secretory pathway (Rubartelli, A, et al., J. Biol. Chem.,267:24161-24164, 1992). The concentrations of Trx found in the medium,up to 10 nm after 6 hr, are lower than those required to directlystimulate cell proliferation (Gasdaska, J. R, et al., Cell Growth &Differ., 6: 1643-1650, 1995). However, we have recently found that Trxat nanomolar concentrations will potentiate the growth effects ofcytokines such as interleukin-2 and basic fibroblast growth factor. Itremains to be established whether the extra trx is producing its effectson cell proliferation through an intracellular or an extracellularaction. It should be noted that Trx binds to the surface of cells(Gasdaska, J. R, et al., Cell Growth & Differ., 6:1643-1650, 1995;Ifversen, P, et al., Hum. Antib. Hybrid, 4:115-123, 1993) so thatsecreted Trx could have a local effect at the outer cell surfacealthough concentrations in the medium are low.

The levels of transfected Trx miRNA in cells were not high, only up to1-fold endogenous trx mRNA levels, and were independent of the mammaliantransfection vector used. Typically mRNA levels resulting fromtransfection using such vectors are 10-50-fold or higher (Powis, G, etal., Anticancer Res., 15:1141-1146, 1995). It may be that high levels ofTrx gene expression are toxic to cells. We have found only a lowexpression of the human Trx gene in transgenic mice. In contrast, somehuman tumors show very high levels of Trx mRNA compared with the normaltissue: more than 11-fold in human primary lung tumors (Gasdaska, P, etal., Proc. Am. Assoc. Cancer Res., 34:62, 1993) and even higher in humanprimary colon tumors (Berggren, M, et al., Anticancer Res.,17:3371-3380, 1997). It is not known why higher trx mRNA levels couldnot be obtained in transfected cells. We were unable to obtaintransformation of NIH 3T3 cells with Trx. It remains to be demonstratedwhether the much higher levels of Trx expression seen in some humantumors might be transforming.

The observation that a redox-inactive dominant-negative Trx reverses thetransformed phenotype of MCF-7 cells suggests that drugs that inhibitthe redox activity of Trx might offer a novel approach to treating someforms of human cancer. Alternatively, inhibiting the enzyme responsiblefor the reduction of trx, the flavoprotein Trx reductase, might alsolead to a selective inhibition of cancer cell growth. We have shownpreviously that some antitumor quinones, including doxorubicin anddiaziquone, are mechanism-based (suicide substrate) inhibitors of Trxreductase both in the purified form and in intact cells (Mau, B. L, etal., Biochem. Pharmacol, 43:1621-1626, 1992). However, the antitumorquinones have many other effects that could contribute to theirantitumor activity (Powis, G. Pharmacol Ther., 35:157-162, 1987). On thebasis of our transfection studies, it would be of great interest to seewhat effect selective inhibitors of Trx or its reductase have on cancercell growth and transformation.

In summary, our results have shown that stable transfection ofnontransformed mouse fibroblast-like cells and human breast cancer cellswith human Trx leads to low levels of overexpression and increased cellsaturation densities but no transformation, measured by tumor formationof NIH 3T3 cells in immunodeficient mice, Stable transfection withredox-inactive mutant Trx results in a dominant-negative effect withinhibition of mouse cell and human breast cancer cell growth andreversion of the transformed phenotype of human breast cancer cells,measured by their ability to form colonies in soft agarose and to formtumors in mice. The Trx produced appears to be secreted mostly fromcells, and whether the Trx is having an intracellular or extracellularaction remains to be determined.

In the majority of the subjects tested ( 8/10), human primary gastriccarcinomas thioredoxin was over-expressed in tumor cells compared tonormal mucosa, and in all cases the over-expression was found only inthe cancer cells and not in stromal cells or infiltrating lymphocytes.Levels of thioredoxin significantly higher than in normal dividingcells, were found in ⅝ of the over-expressing carcinomas. To relatethioredoxin over-expression to cell proliferation and apoptosis, nuclearproliferation antigen was detected by Ki67 antibody and apoptosis by thein situ terminal deoxynucleotidyl transferase (TUNEL) assay weremeasured in the same tissue samples. (See Table 2). Thioredoxinexpression was significantly and highly positively correlatedwith-nuclear proliferation antigen (p<0.01) a marker of aggressive tumorgrowth and highly negatively correlated with apoptosis (p<0.001) a formprogrammed cell death that is presumed to limit tumor growth. Thus,thioredoxin is over-expressed at the mRNA and protein level in a numberof human primary tumors. Further, the expression of thioredoxin proteinis directly associated with highly proliferative tumors.

TABLE 2 Staining of Thioredoxin in Human Gastric Cancers: Comparisonwith Cell Proliferation and Apoptosis Tumor Tumor Thiredoxin ThioredoxinProliferation Apoptosis Patient # Normal* Tumor Ki-67 Tunel 1. ++ +++++++ + 2. ++ +++ +++ + 3. ++ ++++ NE + 4. ++ ++++ ++++ + 5. ++ +++ +++ ++6. ++ 0 + ++++ 7. ++ + + +++ 8. ++ 0 + ++++ 9. ++ ++ +++ +++ 10. ++ ++NE ++ Staining scored as absent (0) or weak (+) to intense (++++) *=gastric pits; NE = non evaluable

MCF-7 human breast cancer cells were transfected with cDNA forthioredoxin or with a catalytic site redox-inactive mutant thioredoxin,C32S/C35S, using two constitutive eukaryotic expression vectors (pRXneoand pDC304neo) and a number of clones were selected for each. The levelof transfected thioredoxin and C32S/C35S thioredoxin mRNA was up to2-fold the endogenous message. Both types of transfected cells showedincreased thioredoxin protein production, measured by quantitativeWestern blotting, up to 100% that of mock-transfected cells.

There was little difference in the growth of the transfected cellsformed up to 4-fold more colonies when grown in soft agarose and theC32S/C35S transfected cells formed up to 80% fewer colonies asillustrated in FIGS. 4A-B.

When these cells were injected into Scid mice the thioredoxintransfected cells formed tumors while the C32S/C335S transfected cellsdid not form tumors, as illustrated in FIG. 5. This was confirmed byhistology. Thus, a dominant-negative redox inactive thioredoxin canreverse the transformed phenotype and inhibits tumor growth in vivoproviding molecular biology evidencing that thioredoxin is a noveltarget for anti-cancer drug development.

4. USE OF THIOREDOXIN AS AN ANTI-TUMOR DRUG TARGET

Although thioredoxin is a known protein, it has not been disclosed orsuggested that thioredoxin be used as a screen for anti-tumor agents. Ithas now been shown that stable transfection of the MCF-7 breast cancercells with a redox-inactive mutant thioredoxin causes inhibition ofanchorage-independent growth of the cells in soft agarose and causescomplete inhibition of tumor formation in vivo. The redox-inactivemutant is formed from thioredoxin where the catalytic site cysteineresidues are replaced with serine. Further, it was shown that the mutantthioredoxin did not inhibit monolayer growth of the cells, i.e., doesnot inhibit normal cell growth, while it causes inhibition ofanchorage-independent growth of the cells in soft agarose, i.e., doesinhibit an in vitro characteristic of tumor cell growth. This is theactivity that would be expected from drugs that inhibit thioredoxin.

a. Examples of Agents that Inhibit Thioredoxin

Agents that inhibit thioredoxin have been identified in accordance withthe present invention, such agents may be antibodies, drugs orantisense. A series of unsymmetrical 2-imidazolyl disulfides wereinvestigated as inhibitors of the thioredoxin system and as potentialanti tumor agents. Although these agents, such as 1-methylpropyl2-imidazolyl disulfide, were originally identified as competitiveinhibitors of thioredoxin reductase (Oblong J E, et al., CancerChemother. Pharmacol, 34:434-438, 1994) but it has now been shown thatthey also to bind irreversibly to Cys⁷³ of thioredoxin and to block itsreduction by thioredoxin reductase. A number of these disulfidecompounds have been studied and have demonstrated anti-tumor activityagainst human tumor xenografts in Scid mice with up to 90% inhibition ofMCF-7 breast cancer and HL-60 promyelocytic leukemia growth. It has nowbeen demonstrated that the imidazolyl disulfides inhibitthioredoxin-dependent cell growth (Oblong J E, et al., Cancer Chemother.Pharmacol, 34:434-438, 1994) and that their growth inhibitory activityin the National Cancer Institute 60 human tumor cell line panelcorrelates with levels of thioredoxin mRNA in these cell lines (BerggrenM, et al., Anticancer Res., 16:3459-3466, 1996), A COMPARE correlativeanalysis of the activity of the lead disulfide compounds in the NCI cellline panel with over 50,000 compounds already tested for cell growthinhibition by the NCI was conducted in order to identify compounds witha similar pattern of growth inhibitory activity. Some of the compoundsidentified in this way were inhibitor of thioredoxin reductase and somewere inhibitors of thioredoxin.

5. USE OF THIOREDOXIN REDUCTASE AS A TARGET FOR INDUCINGANTI-PROLIFERATION

Although the general properties of human thioredoxin reductase as aprotein and the cDNA base sequence of human thioredoxin reductase hasbeen known in the art, it has now been discovered that thioredoxinreductase is useful as an anti-cancer drug target. It has now been shownabove that redox activity is necessary for the growth stimulatingactivity of thioredoxin. Since thioredoxin reductase is the only knownway for thioredoxin to be reduced biologically it is an obviousextension of the above observations that thioredoxin reductase couldalso be a target for the development of anti-cancer drugs.

6. USE OF RECOMBINANT MODIFIED THIOREDOXIN FOR STIMULATING CELL GROWTH

It has been discovered that human thioredoxin, and specificallyrecombinant modified thioredoxin (mutated thioredoxin), does not undergospontaneous oxidation and/or dimer formation, or protected againstbreakdown by blood and tissues, may have therapeutic utility insituations where stimulation of cell growth is preferred or required. Ina non-limiting embodiment of the present invention, such new usesinclude, and are not limited to, the beneficial use of thioredoxinand/or recombinant modified thioredoxin in stimulating cellproliferation in individuals (1) with myelodysplastic syndrome; (2) inneed of bone marrow transplantation; (3) with post-chemotherapy tostimulate bone marrow growth; (4) in need of stimulation of the immunesystem; (5) in need of stimulation of wound healing; (6) such astransgenic animals in need of stimulation of body growth; (7) in need ofsimulation of the responses to cytokines and growth factors for growthstimulation effects; and (8) in gene therapy techniques.

The underlying defect in myelodysplastic syndrome is decreasedmultilineage progenitor cell growth associated with decreasedsensitivity to growth factor stimulation. Thioredoxin acts to increasethe sensitivity of cells to growth factors and stimulates multilineageprogenitor cells which provides a beneficial utility in individuals withMDS.

In individuals in need of bone marrow transplantation, it would be ofgreat utility to promote the growth of transplanted cells. Thioredoxinmay be used to protect hematopoietic progenitor cells and to expandcells ex vivo for bone marrow cell growth. This would rely on aselective effect for bone marrow since tumor cells might also bestimulated by the thioredoxin.

It would provide a great benefit to individuals subject to chemotherapytreatment to selectively stimulate bone marrow cell growthpost-chemotherapy.

It has been found that Cys73->Ser mutant thioredoxin will stimulate theproliferation of human immune cells in culture, which can provide agreat benefit to individuals in need of stimulation of immune systemcells.

Wild-type and Cys73->Ser mutant thioredoxin also stimulates the growthof fibroblasts, which are important components of wound healing process.There would be a great advantage of using thioredoxins to stimulatewound healing, for example after surgery.

It has been found that wild-type thioredoxin expressed as a transgene inmice may be lethal or is only weakly expressed. Therefore, it ispossible that construction of transgenes with wild-type or mutant formsof thioredoxin, with or without tissue specific and/or induciblepromoters, could be used to stimulate the development of the animal orthe growth of selected organs.

It has been found that thioredoxin can potentiate the response of cellsin culture to growth factors and cytokines such as IL-2 and basicfibroblast growth factor (bFGF). Combinations of thioredoxin with othergrowth factors or cytokines therefore increases the therapeuticusefulness of these growth factors where increased cell proliferation isthe desired therapeutic effect.

Introduction of the thioredoxin or mutant thioredoxin genes into humancells provides a mechanism of improving the therapeutic usefulness ofother cytokines or growth factors given directly or themselves as genetherapy, for example IL-2.

a. Examples of Stimulation of Cell Growth Using Thioredoxin Protein

Although thioredoxin mRNA has been found to be over expressed by somehuman tumor cells, it has been discovered that thioredoxin, specificallyrecombinant modified thioredoxin also stimulates cell growth.

A novel mechanism or over-expression and secretion from the cells by aleaderless secretory pathway has important consequences for potentialtherapeutic uses of thioredoxin as explained below. It has beendiscovered that human recombinant thioredoxin undergoes spontaneousoxidation in air to give a form that will not stimulate cell growth.This spontaneous oxidation appears to involves Cys73 since a mutantthioredoxin where this residue has been converted to serine (Cys73->Serthioredoxin) does not undergo this loss of activity. X-raycrystallography studies of wild-type and C73S thioredoxin show thatthioredoxin has a highly conserved hydrophobic dimer forming surface andthat Cys73 stabilizes homodimer formation through a Cys73-Cys73disulfide bond (Weichsel A, et al. Structure 4:735-751 (1996)). Theactive site Cys residues become relatively inaccessible in thethioredoxin homodimer so that it is a very poor substrate forthioredoxin reductase. The thioredoxin homodimer does not stimulate cellproliferation. The half life of recombinant human thioredoxin inphosphate buffered 0.9% NaCI at −20° C. is 6-8 days. Thus, the wild typethioredoxin is not a good protein for therapeutic use because of istendency to oxidize and lose biological activity.

It has been found that wild-type thioredoxin loses its ability tostimulate cell proliferation even over a few days even before formationof the Cys73-Cys73 disulfide stabilized dimer. This appears to be due tomodification of the monomeric form of thioredoxin possibly involvingreversible dimerization without covalent linkage, or to other oxidativeevents in the protein. In contrast, it has been found that Cys73->Serthioredoxin is stable in solution over several weeks, even at roomtemperature, and does not dimerize. Cys73->Ser thioredoxin is aseffective as wild-type thioredoxin at stimulating cell proliferation andretains this ability with no loss over many days and, thus, appears tobe more suitable as a potential therapeutic agent.

In order to investigate whether thioredoxin and mutant thioredoxinproteins have activity in intact animals I studied the ability of theCys73->Ser mutant thioredoxin to prolong the life of mice that had beenlethally y-irradiated and which, if untreated, die from bone marrowsuppression as shown in Table 3. Mice that had been injected with theCys73->Ser mutant thioredoxin survived 850 Gy y-radiation whereasnon-injected mice died. Thus, Cys7->Ser mutant thioredoxin can preventthe death of lethally y-irradiated mice. While not wishing to be boundto any particular theory, it is presumed that this effect is due tostimulation of bone marrow cell growth.

TABLE 3 Protection Against Radiation Induced Death by Cys73−>Ser MutantThioredoxin Cys72−>Ser thioredoxin mouse control day of death day ofdeath 1 11 alive 2 16 alive 3 16 alive 4 16 alive 5 17 alive median 1.6± 2.1 alive Mice received 8.5 Gy y-irradiation. One group of mice wastreated with Cys73−>Ser thioredoxin in 0.9% NaCl 0.85 mg/mouse injectedi.v. 30 min before and 24 hr. after radiation. There were 6 mice m thecontrol group and 4 mice in the treated group. The study was terminatedon day 30.

Evidence that Cys73->Ser mutant Thioredoxin stimulates the growth ofbone marrow was obtained directly by adding Cys⁷³->Ser mutantthioredoxin directly to human bone marrow and studying its effects oncolony formation by the cells, as illustrated in FIG. 1. Cys73->Sermutant thioredoxin stimulates colony formation by the multilineageprogenitor cells (CFU-GEMM) but does not stimulate the lineage specificerythroid progenitor (BFU-E) and myeloid progenitor (CFU-GM) cells.

1 illustrates the stimulation of human bone marrow colony formation byCys73->Ser mutant thioredoxin, in accordance with the present invention.Human bone marrow was obtained as excess material from normal allogeneicbone marrow donors. The effects of Cys⁷³->Ser thioredoxin on colonyformation are shown by (o) multilineage progenitors (CFU-GEMM); (•)erythroid progenitors (BFU-E); and (∇) myeloid progenitors (CFU-GM), asmeasured over 10 days as described. (Values are the mean of 4determinations and bars are S.D.)

It has further been found that Cys73->Ser thioredoxin can stimulate cellproliferation by increasing the response of the cells to other cytokinesor growth factors such as interleukin-2 (IL-2) and fibroblast growthfactor (FGF) as illustrated in the chart in FIG. 2. FIG. 2 illustratespotentiation of IL-2 induced MCF-7 breast cancer cell growth byCys73->Ser mutant thioredoxin, in accordance with the present invention.Cells were growth arrested for 48 hr, in medium with 0.5% serum (10^(s)cells) then stimulated in the absence of medium with either IL-2 orCys32->Ser mutant thioredoxin at the concentrations shown, Cell numberwas measured after 48 hr. Each point on the chart represents the mean of3 determinations and bars represent S.E. The dotted line showsstimulation by 10% serum.

In addition, antibodies to the receptors for the growth factors canblock the response to these agents, in accordance with the presentinvention as shown in FIG. 3. FIG. 3 illustrates the inhibition ofthioredoxin stimulated MCF-7 cell growth by receptor antibodies, inaccordance with the present invention. Cell proliferation was measuredas described above in the context of FIG. 2. The concentrations ofagents used were Cys73->Ser mutant thioredoxin (Thioredoxin) 1 μM;monoclonal antibodies to FGF receptor, IL-2-receptor and EGF-receptor 4μg/ml; and EGF 10 nM. The EGF and EGFR were added as a negative control.Values represent the mean of 3 determinations and bars represent S.E.The dotted line shows the effect of 10% serum alone.

Therefore, the discovery that human thioredoxin, and specificallyrecombinant modified thioredoxin, does not undergo spontaneous oxidationand/or dimer formation has a tremendous potential in vivo utility insituations where stimulation of cell growth is required. In addition, itmay be advantageous to modify the thioredoxin structure to increase thepotency and therapeutic usefulness, such as changing the amino acidsequence at the site of proteolytic cleavage to prevent breakdown byplasma enzymes.

Thioredoxin/mutant thioredoxin may have use after bone marrowtransplantation of cancer patients or together with chemotherapy tostimulate bone marrow recovery, or to stimulate the immune system inpatients with AIDS. There may be other potential therapeuticapplications for thioredoxin/mutant thioredoxin such as increasing therate of wound healing. If a thioredoxin or mutant thioredoxin gene couldbe introduced into an animal as a transgene this might result in anincreased growth rate of the animal. A thioredoxin transgenic mouse hasbeen developed, although the levels of gene expression are very low andthe animal does not show an increased growth rate. However, a gene formutant thioredoxin might be more effective in this regard. The use ofmutant thioredoxins may not be limited to the Cys73->Ser mutant.Mutation of the other Cys residues can also affect biological activity.There are also other amino acid residues on the hydrophobic domain ofthe molecule that X-ray crystallographic studies have shown might alsobe important for dimer formation. Mutation of these and possibly otheramino acid residues, might alter the biological activity of thioredoxin.

The in vitro cell growth stimulating activity of human thioredoxin hasbeen previously reported for human lymphoid and solid tumor cancer cells(Gasdaska J R., et al., Cell Growth Differ., 6:1643-1650, 1995; Oblong JE, et al., J. Biol. Chem., 269:11714-11720, 1994) and for mousefibroblast cells (Oblong J E, et al., J. Biol. Chem., 269:11714-11720,1994). The production of a Cys73->Ser mutant thioredoxin has beenpreviously reported. In one study it did not act like wild-typethioredoxin as a component of a complex cell growth stimulating factorcalled “early pregnancy factor” (Tonissen K, et al., J. Biol. Chem.,268:22485-22489, 1993). In another study it was reported that Cys73->Sermutant thioredoxin did not form a dimer, but cell growth stimulatingactivity by the mutant thioredoxin was not investigated in this study(Ren X, et al, Biochem., 32:9701-9705, 1993). However, the ability ofthe Cys73->Ser mutant and other mutant thioredoxins to stimulate cellproliferation has not been reported. There have been no prior reports ofadministration of wild-type or mutant thioredoxins in vivo.

b. Role of Oxidative Inactivation of Thioredoxin as a Cellular GrowthFactor

Thioredoxin (Trx) is a widely distributed redox protein that regulatesseveral intracellular redox-dependent processes and stimulates theproliferation of both normal and tumor cells. We have found that whenstored in the absence of reducing agents, human recombinant Trxundergoes spontaneous oxidation, losing its ability to stimulate cellgrowth, but is still a substrate for NADPH-dependent reduction by humanthioredoxin reductase. There is a slower spontaneous conversion of Trxto a homodimer that is not a substrate for reduction by thioredoxinreductase and that does not stimulate cell proliferation. Bothconversions can be induced by chemical oxidants and are reversible bytreatment with the thiol reducing agent—dithiothreitol. SDS-PAGEsuggests that Trx undergoes oxidation to monomeric form(s) precedingdimer formation. We have recently shown by X-ray crystallography thatTrx forms a dimer that is stabilized by an intermolecular Cys⁷³-Cys⁷³disulfide bond. A Cys⁷³->Ser mutant Trx (C73S) was prepared to determinethe role of Cys⁷³ in oxidative stability and growth stimulation. C73Swas as effective as Trx in stimulating cell growth and was a comparablesubstrate for thioredoxin reductase. C73S did not show spontaneous oroxidant-induced loss of activity and did not fort a dimer. The resultssuggest that Trx can exist in monomeric forms, some of which aremediated by Cys⁷³ that do not stimulate cell proliferation but can bereduced by thioredoxin reductase. Cys⁷³ is also involved in formation ofan enzymatically inactive homodimer, which occurs on long term, storageor by chemical oxidation. Thus, although clearly involved in proteininactivation, Cys⁷³ is not necessary for the growth stimulating activityof Trx.

Trx is a redox protein found in both eukaryotes and prokaryotes(Holmgren A., Annu. Rev. Biochem., 54:237-271, 1985). The redox activityof Trx arises from a highly conserved Trp-Cys-Gly-Pro-Cys-Lys activesite sequence where the 2 cysteine residues (Cys) undergo reversibleoxidation to cystine. Reduction of Trx is catalyzed by thioredoxinreductase (Luthman M, et al., Biochem., 21:6628-6633, 1982). Trx wasoriginally identified in Escherichia coli as a hydrogen donor forribonucleotide reductase (Laurent T C, et al., J. Biol. Chem.,239:3436-3444, 1964). Trx has since been found to act as anintracellular dithiol-disulfide reductase and to modulate the activityof a number of intracellular proteins (Fountoulakis M., J. Biol. Chem.,267:7095-7100, 1992; Kistner 25 A. et al., Toxicon., 31:1423-1434, 1993;Silverman R B et al., Biochem. Biophys. Res. Commun., 155:1248-1254,1988) including the DNA binding of transcription factors (Hayashi T, etal., J. Biol. Chem., 268:11380-11388, 1993; Gaiter D, et al., Eur. J.Biochem., 221:639-648. 1994; Grippo J F et al., J. Biol. Chem.,258:13658-13664, 1983; Cromlish J A et al., J. Biol. Chem.,264:1.8100-18109, 1989). Trx-like sequences are found in other proteinsincluding protein disulfide isomerase (Freedman R B et al., TrendsBiochem. ScL., 19:331-336, 1994). There is evidence that Trx may play arole in the growth and transformed phenotype of some cancers. Trx isover expressed by a number of human cancers compared with normal tissue(Berggren M, et al., Anticancer Res. 17:3377-3380, 1997); Gasdaska P Yet al., Biochem. Biophys. Acta, 1218:292-296, 1994, Nakamura H, et al.,Cancer 69:2091-2097, 1992). It has recently been shown that transfectionof human cancer cells with, a dominant-negative mutant human Trxinhibits anchorage-independent growth in vitro and tumor formation invivo (Gallegos A, et al., Cancer Res. 56:5765-5770, 1996).

As well as having intracellular actions, Trx acts exogenously as aredox-active growth factor. Human Trx is identical to the leukemic cellautocrine growth factor adult T-cell leukemic factor (Gasdaska P Y etal., Biochem. Biophys. Ada., 1218:292-296, 1994), and stimulates thegrowth of both normal fibroblasts (Oblong J E et al., J. Biol. Chem.,269:11714-11720, 1994) and human hematologic and solid tumor cancercells in culture (Wakasugi N, et al. Proc. Natl. Acad. Sci USA,87:8282-8286, 1990; Gasdaska J R et al., Cell Growth Differ.,6:1643-1650, 1995). Trx appears to act by a helper mechanism thatsensitizes the cells to growth factors secreted by the cells themselves(Gasdaska J R et al., Cell Growth Differ, 6:1643-1650, 1995). Mutanthuman Trxs, where the Cys³² and Cys³⁵ residues at the catalytic site(numbering of amino acid residues is from the N-terminal methionine,although this may be removed in some forms of Trx) are converted toserines (Ser) either singly or together, are redox inactive and do notstimulate cell growth (Oblong J E, et al., J. Biol. Chem.,269:11714-11720, 1994). Trx is secreted from cells by a leaderlesssecretory pathway (Rubartelli A, et al., J. Biol. Chem.,267:24161-24164, 1992) and could be acting as an autocrine factor forthe growth of some cancer cells (Gasdaska J R, et al., Cell GrowthDiffer., 6:1643-1650, 1995).

We have found that E. coli Trx, unlike human Trx, does not stimulate thegrowth of human solid cancer cells (Gasdaska J R, et al., Cell GrowthDiffer., 6:1643-1650, 1995). The structures of E. coli and human Trx aresimilar, and both are substrates for human thioredoxin reductase.However, the surface residues of the two forms vary considerably(Weichsel A, et al., Structure 4:735-751, 1996). Human Trx has 3additional cysteine residues, Cys⁶², Cys⁶⁹ and Cys⁷³, in addition tothose in the active site, which do not normally form intramoleculardisulfide bonds (Weichsel A, et al., Structure 4:735-751, 1996;Forman-Kay J D et al., Biochem., 30:2685-2698, 1991). Trx can also forma homodimer with al 100 A² interface domain and a disulfide bond betweenCys⁷³ from each monomer (Weichsel A, et al., Structure 4:735-751, 1996).During our studies of cell growth stimulation by Trx we observed thatstorage of the Trx without a reducing agent for even a few days resultedin a loss of its growth-stimulating activity, although the Trx remaineda substrate for reduction by thioredoxin reductase. We have, therefore,examined the, role of spontaneous and induced oxidation of Trx andcysteine-deleted mutant Trxs, and their ability to stimulate cellproliferation.

(i) Preparation of Thioredoxins

Recombinant human Trx and Cys³²->Ser/Cys³⁵->Ser mutant Trx (C32S/C35S)were prepared and purified as previously described (Oblong J E et al.,J. Biol. Chem., 269:11714-11720, 1994). Cys⁷³->Ser mutant human Trx(C73S) was prepared from single-stranded, sense strand human Trx cDNAligated by polyethylene glycol precipitation into the pBluescript KSvector (Stratagene, La Jolla, Calif.) using helper phage. Thesingle-stranded cDNA was used for oligonucleotide-directed in vitromutagenesis (Version 2.1 Kit, Amersham, Buckinghamshire, U.K.) usingoligonucleotide 5′-TGTTGCATGGATTTGACTTC-3′. Point mutagenesis wasconfirmed by dideoxy sequencing of base-denatured double-stranded. DNAusing the Sequenase Version 2.0 kit (USB, Cleveland, Ohio). Novel Nde1and BamH1 restricted sites were introduced at the 5′ and 3′ ends of themutant Trx cDNA by oligonucleotide-directed PCR. Ndel/BamHI restrictedfragments were extracted from agarose gels, ligated into a suitablyrestricted pET-3a expression vector (Studier F W et al., MethodsEnzymol., 185:60-89, 1991), transformed into E. coli BL21 cells andconfirmed by dideoxy sequencing. C73S Trx was expressed and purified aspreviously described (Oblong J E et al., J. Biol. Chem.,269:11714-11720, 1994). All Trxs were stored at −20° as a 25 μM stocksolution in 5 mM DTT. Before use, the DTT was removed by passing the Trxsolution through a PD-10 desalting column (Pharmacia, Uppsala, Sweden).The Trx solution was kept at 4° and used within 2 hr. (fresh) or storedin water or 0.1 M potassium phosphate-buffered 0.9% NaCI at 4° or −20°for specified times. Oxidized Trx for cell growth studies was preparedby adding a 5-fold molar excess of H₂O₂, to a 25 μm Trx stock solutionwithout DTT and 1 hr. later removing unrecalled H₂O₂ using a PD-10column.

(ii) Cell Growth Studies

MCF-7 human breast cancer cells were obtained from the American TypeCulture Collection (Rockville, Md.) maintained in DMEM containing 10%fetal bovine serum at 37° and 6% CO₂, and passaged at 75% confluence.The effect of Trx and modified Trxs on MCF-7 cell growth was determinedas previously described (Gasdaska J R et al., Cell Growth Differ.,6:1643-1650, 1995). Briefly, 105 cells were plated in a 35-mm culturedish in DMEM containing 10% fetal bovine serum and, after attachment for24 hr., growth arrested using DMEM with 0.5% fetal bovine serum for 48hr. The medium was then replaced with DMEM containing Trx or otheradditions for 2 days and cell number measured with a hemocytometer. Allincubations were conducted in triplicate.

(iii) Thioredoxin Reductase Assay

Human placenta thioredoxin reductase, specific activity 33.3 μmol Trxreduced/min/mg protein, was prepared as previously described (Oblong J Eet al., Biochem., 32:7271-7277, 1993). Reduction of Trx and C73S bythioredoxin reductase was measured by the oxidation of NADPH at 340 nMwith insulin as the final electron acceptor as described by Luthman andHolmgren (Luthman M, et al., Biochem., 21:6628-6633, 1982).

(iv) Electrophoresis

A 25 μM solution of fresh Trx, mutant C73S or C32S/C35S Trxs, Trxs thathad been aged at room temperature for 48 hr., 7 days, 90 days; or Trxstreated for 1 hr. with 1 mM diamide, 10 mM DTT, 3 mM 2-mercaptoethanolor 2:1 (v:v) H₂O₂, was mixed with an equal volume of loading buffercontaining 3% SDS, 10% glycerol and 0.1% bromophenol blue in 0.05 MTris-HCI, pH 6.8. Approximately 0.02 ng of the protein was 30 loaded ineach lane of a 24×45 cm 16.5% polyacrylamide resolving gel (pH 8.4)containing 0.3% SDS, a 10% spacer gel and a 6% stacking gel andseparated by electrophoresis using an anode buffer of 0.2M Tris-HCI, pH8.9 and cathode buffer of 0.1 M Tris-HCI, 0.1% SDS, pH 8.2. The gel wasrun for 1. hr. at 400 volts before loading the samples and then at 400volts for 24 hr. before fixing in 50% methanol, 7.5% acetic acid for 20min, followed by 5% methanol, 7.5% acetic acid for 20 min, followed by10% glutaraldehyde for 20 min. The gel was soaked in 2 L H₂O overnightto remove unbound SDS and then silver stained (ICN Silver Stain Kit,Irvine, Calif.), Similar observations were made when the gels werestained with Coomassie Blue.

(v) Growth Stimulation

Cys⁷³->Ser mutant Trx (C73S) stimulated the proliferation of human MCF-7breast cancer cells. The EC₅₀ for growth stimulation by C73S was 350 nMand the maximum effect was seen at 1 μM, which is similar to values wehave previously reported for stimulation of MCF-7 cell proliferation byrecombinant human Trx (Gasdaska J R et al., Cell Growth Differ6:1643-1650, 1995). Storage of Trx in the absence of a reducing agentsuch as DTT at 40 for 5 days resulted in a 78% loss, and for 90 days a98% loss of cell growth stimulating activity (FIG. 13). In contrast C37Sshowed no loss of activity when stored under these conditions. Trxstored in the presence of bovine catalase at 1 unit/ml did not losebiological activity over a 5-day period (results not shown).

(i) Reduction of Thioredoxin by Thioredoxin Reductase

C73S was a good substrate for reduction by human placental thioredoxinreductase with a K_(m) of 0.20 μM and a V_(max) of 6.3 nmol/min/μg.These values are similar to those we have previously found for freshTrx, which were a K_(m) of 0.33 μM and a V_(max) of 5.9 nmol/min/μg(Oblong J E et al., Biochem., 32:7271-7277, 1993).

The effect of storing Trx without DTT on its ability to act as asubstrate for thioredoxin reductase was investigated (Table 4). Whenstored in H₂O either at −20° or at room temperature Trx showed a loss ofactivity with a half-life of 20-30-days. The loss of Trx activity wasmore rapid when stored in phosphate-buffered 0.9% NaCl, with a half lifeof 8 days. Phosphate buffer is known to contain small amounts of iron(Poyer J L et al., J. Biol. Chem., 246:263-269, 1971), which couldcatalyze an oxidative process increasing the loss of Trx activity.Alternatively, the lower pH of the solution in water could stabilize Trxor the increase in ionic strength of phosphate-buffered 0.9% NaCl couldenhance the formation of the inactive homodimer of Trx. The aged Trxshowed a slow, delayed reduction by thioredoxin reductase that wasstimulated by catalytic amounts of fresh Trx (FIG. 14). It is importantto note that the loss of activity of Trx as a substrate for thioredoxinreductase was much slower than the loss of activity as a stimulator ofcell growth. C73S did not show a loss of activity as substrate forthioredoxin reductase upon storage for up to 30 days. The ability of Trxto act as a substrate for thioredoxin reductase was completely inhibitedby treatment with 5 molar equivalents of H₂O₂, whereas C37S remainedfully active after treatment with 100 molar equivalents of H₂O₂ (FIG.15).

TABLE 4 Effect of Storage of Thioredoxins As Substrates for ThioredoxinReductase InH20 In PBS −20° +21° −20° −21° t½(days) t½(days) t½ (days)t½ (days) Trx 30.5 20.1 8.2 7_8 C73S stable* stable* stable* stable* 25μMstock solution of Trx of C73S in H₂O or phosphate buffered 0.9% NaCl(PBS) free of D77 were stored frozen at −20° or at room temperature(+21°) for up to 60 days and their ability to act as a substrate forreduction by human placental thioredoxin was measured. A first orderdecrease in activity was found for Trx and the results are expressed ashalf-life *<10% loss of activity over 30 days.

(vii) Multiple Forms of Thioredoxin

Electrophoretic analysis of freshly prepared human Trx stored in DTTshowed a mixture of 5 bands of apparent molecular weights ranging from8.1 to 11 kDa (FIGS. 16, 17, and 18, lane 1). Storage of Trx at roomtemperature without DTT resulted in a change in the banding pattern withdisappearance of the 8.1-kDa band by 48 hr. (FIG. 16, lane 2). Storageof Trx without DTT for 7 days resulted in the loss of additional bandsand the appearance of a new band at 23 kDa due, apparently, to a Trxdimer (FIG. 16, lane 3). Storage of Trx without DTT for 90 days at 4°resulted in almost complete conversion to the Trx dimer (FIG. 16, lane4). Treatment of 7-day aged Trx (FIG. 17, lane 2) with 2-mercaptoethanolresulted in the reappearance of the fresh Trx banding pattern, exceptfor the 8.1-kDa band, which did not reappear (FIG. 17, lane 3). Loss ofthe smaller bands and dimer formation was seen when Trx was treated withdiamide, a protein thiol oxidizing agent (Kosower N S et al., Methods.Enzymol, 251:123-132, 1995) (FIG. 17, lane 5). The formation of Trxdimer following diamide treatment was also confirmed by gel permeationchromatography (results are not shown). H₂O₂ treatment of Trx alsocaused dimerization but produced a different banding pattern to thatproduced by diamide (FIG. 17, lane 6). Treatment of Trx with NEM, athiol alkylating agent (Gilbert H F, Methods Enzymol., 251:8-30, 1995),gave a single band with a slightly elevated apparent molecular weight,but no dimer formation (FIG. 17, lane 4). Treatment of 7 day aged Trxwith NEM produced both the higher molecular weight band as in FIG. 17,lane 4, and the bands illustrated in FIG. 17, lane 2 (data not shown),suggesting that in the aged Trx not all the sulfhydryls are availablefor covalent modification. None of the changes caused by NEM werereversed with 2-mercaptoethanol treatment (data not shown).2-Mercaptoethanol reversed Trx dimer formation caused by both diamideand H₂O₂, treatment (FIG. 17, lanes 7 and 8) but was less effective atreversing changes in the monomeric banding pattern of Trx produced byH₂O₂, (FIG. 17, lane 8).

Freshly prepared C73S Trx and C32S/C35S Trx showed fewer bands than wildtype Trx (FIG. 18, lanes 2 and 3, compared with lane 1). Treatment ofC32S/C35S Trx with diamide resulted in the formation of a 23-kDa dimer(FIG. 18, lane 6). Treatment of C7¹)S Trx with diamide caused the bandsto coalesce into a single band of around 10 kDa, but there was no 23-kDadimer formed (FIG. 18, lane 4). The effects of diamide on C37S and02S/05S were reversed by treatment with DTT (FIG. 18, lanes 5 and 7).

(viii) Discussion

The study shows that human recombinant Trx undergoes at least 2 levelsof spontaneous and induced oxidative transformation. The first oxidationoccurs spontaneously within a few days to a form(s) that can no longerstimulate cell growth but remains a substrate for thioredoxin reductase.The slower oxidation, occurs over a period of weeks, or can be inducedby the thiol oxidizing agent diamide, and leads to a disulfide bandedhomodimer which not only fails to stimulate cell growth but is a poorsubstrate for thioredoxin reductase. The fact that similar changes inTrx can be induced by chemical oxidation, are protected against bycatalase and are reversed by the thiol reducing agent DTT is consistentwith the interpretation that the changes in Trx are due to oxidation.Cys⁷³ appears to play a critical role in both levels of oxidant-inducedinactivation since C73S does not lose the biological activity or itsability to act as a substrate for thioredoxin reductase upon aging.

We have shown by SDS-PAGE that fresh human recombinant Trx can exist inat least five different states, which probably reflect the fully reducedstate of the protein as well as different intramolecular disulfidebonded states due to the five cysteine residues present in the protein.While the specific nature of these intramolecular disulfide bonds is notknown, it is likely that some, at least, are due to non-naturaldisulfide bonded structures which form during denaturation and theoxidizing conditions of extended electrophoresis (Creighton T E, MethodsEnzymol, 107:305-329, 1984). The observation that C317S and C32S/C35Sexhibit a simpler banding pattern than wild-type Trx upon SDS-PAGE alsosuggests that the banding pattern is due to disulfide bond formation.X-ray structural analysis indicates that in addition to a disulfide bondbetween Cys³² and Cys³⁵, the only other intramolecular disulfide bondthat could form in the nondenatured Trx is between Cys⁷³ and Cys³²although even this would require a different conformation of the protein(Weichsel A, et al., Structure 4:735-75.1, 1996). With the exception ofa possible slight modification in Cys⁶⁹ there is no evidence that Cys³²,Cys³⁵ or Cys⁶² are oxidized in Trx crystals formed in the presence of 5mM DTT (Weichsel A, et al., Structure 4:735-751, 1996). The fact thattreatment of Trx with NEM produces only one band implies that prior todenaturation and electrophoresis fresh Trx exists as a single species.The number of free thiols in fresh Trx was determined to be 4.5 to4.6/molecule by Ellman's reagent (Ellman G L, Arch. Biochem. Biophys.,82:70-77, 1959) (data not shown), indicating that all five cysteines arein the sulfhydryl form. Treatment of NEM-alkylated Trx with oxidizing orreducing agents produces no change in the banding pattern (data notshown), which is further evidence that all 5 sulfhydryls have beenalkylated. Oxidation of cysteines to sulfenic or sulfinic acids isunlikely to occur spontaneously (Claiborne A et al., FASEB J7:1483-1490, 1993). It is noteworthy that H₂O₂ treatment of Trx givesrise to a different monomeric banding pattern than that of spontaneouslyoxidized Trx. The original monomeric banding pattern is also notregenerated by treatment with DTT. As has been previously suggested forNADH peroxidase (Poole L B et al., J. Biol. Chem., 264:12330-12338,1989), we speculate that H₂O₂ oxidizes the cysteines to sulfenic acidsand to the irreversible sulfinic or sulfonic acid states.

During the same time interval that there was a loss of the growthstimulating activity of Trx, there was a shift of the electrophoreticbanding pattern. There was a collapse of the banding pattern with lossof some of the Trx monomeric bands over 7 days, suggesting that Trx maybe undergoing “native” intramolecular disulfide bond formation prior toelectrophoresis, which prevents the formation of random disulfidebondformation seen with denaturation and electrophoresis of fresh Trx. Asimilar phenomena has been observed with bovine pancreatic trypsininhibitor (Creighton T E, Methods Enzymol., 107:305-329, 1984; WeissmanJ S, et al., Science 253:1386-1393, 1995). Alleviation of aged Trx withNEM gave more than one protein product, indicating that aged Trx existsin multiple forms and not all the sulfhydryls are available forreaction. Since C73S does not undergo a similar shift in banding patternand does not undergo loss of growth stimulating activity, it can beassumed that Cys⁷³ is involved in this intramolecular disulfide bondformation, perhaps with Cys³² (FIG. 19). Thus, spontaneous aging of Trxover a few days results in the inability of Trx to stimulate cellgrowth, although Trx is still a substrate for reduction by thioredoxinreductase. Analysis of the X-ray structure of Trx shows that Cys⁷³ is byfar the most accessible cysteine residue and possibly the most reactive(Page D L, Am. J. Surg. Pathol, 15:334-349, 1991).

If a solution of Trx is left long enough, or upon treatment with astrong oxidizing agent such as diamide or H₂O₂, there is formation of a23-kDa Trx homodimer. Reduction of the Trx dimer by thioredoxinreductase is slow and delayed, and is stimulated by low concentrationsof fresh Trx, suggesting there may be an autocatalytic process. Asimilar conclusion was reached by Ren X et al., Biochem., 32:9701-9705,1993, Formation of the Trx homodimer appears to involve the Cys⁷³residue since C73S, where Cys⁷³ is replaced with serine, does notundergo oxidation-induced homodimer formation as do Trx and C32S/C35S.Ren X et al., Biochem., 32:9701-9705, 1993 have shown C73S does notundergo oxidative homodimer formation induced byselenodithioglutathione. We recently reported the X-ray crystalstructure of Trx and identified a largely hydrophobic dimer forminginterface that is stabilized by a Cys⁷³-Cys⁷³ disulfide bond (WeichselA, et al., Structure 4:735-751, 1996). Our observation that Trxundergoes a faster loss of activity with thioredoxin reductase in PBSversus water indicates that iron-induced oxidation or an increase inionic strength may stabilize and enhance dimer formation, which isconsistent with the hydrophobic nature of the dimer interface observedin crystals of human Trx.

The importance of the monomeric oxidative form(s) of Trx is unknown.While the structural nature is yet to be identified, it does havedifferent biological activity in our in vitro system. Trx is secreted bycells into the extracellular environment, which is predominantlyoxidizing, and might be expected to undergo monomeric oxidation.Considering its ease of formation, it is reasonable to assume thatmonomeric oxidation will precede oxidative homodimer formation. Whetherthis might be sufficient to prevent Trx from acting as a growth factoris not known. The formation of the oxidized monomer inside the cell isless likely since it still can be slowly reduced by thioredoxinreductase and the interior of the cell is highly reducing.

The physiological significance of homodimer formation is also unknown.What might be Trx homodimer has been reported in diamide-treated Jurkatcells (Sato N, et al., J. Immunol., 154:3194-3203, 1995). I haveobserved small amounts of the Trx homodimer by immunoblotting ofuntreated MCF-7 breast cancer and other cell lysates. It is intriguingto speculate that formation of an oxidized Trx monomer or homodimer inresponse to intracellular oxidants such as H₂O₂ might be a way mammaliancells detect oxidant formation. Trx is believed to exist in normal cellsat concentrations from 1 to 10 μM (Luthman M, et al., Biochem.,21:6628-6633, 1982; Berggren M, et al., Anticancer Res. (in press)),though in selected tissues and specific cell compartments this valuecould be much higher. It is therefore not unreasonable to assume thatTrx will undergo homodimer formation in vivo. As I observed with theenhanced inactivation of Trx in phosphate buffered saline, I expectdimer formation to precede faster in vivo than observed in vitro inwater. Whether dimer formation in vivo would prevent the fasteroxidation to an intramolecular form is unknown. The slow autocatalyticreduction of the Trx homodimer to the monomer would be a way to restorethe cell to normal operating conditions after the induction of oxidativestress.

In summary, we have found that human recombinant Trx undergoesrelatively rapid (over a few days) spontaneous and oxidant-inducedconversion to a form(s) that does not stimulate cell proliferation, butis still a substrate for reduction by thioredoxin reductase. There ismuch slower (over a period of weeks) spontaneous oxidation of Trx to aCys73-stabilized homodimer form that is not a substrate for thioredoxinreductase and that also does not stimulate cell proliferation. Bothconversions can be reversed by treatment with the thiol reducing agentDTT, and both appear to involve the Cys⁷³ residue. A Cys73->Ser mutantTrx, which stimulates cell proliferation and is as effective a substratefor thioredoxin reductase as Trx, did not show age or oxidation-inducedloss of these activities. Thus, with time Trx gradually loses itsability to stimulate cell proliferation and to be a substrate forthioredoxin reductase, unlike the Cys⁷³->Ser mutant Trx, which retainsthese activities with no loss. Thus, Cys⁷³ is not critical forbiological activity but may play a critical role in the oxidativeregulation of Trx activity.

7. REDUCING INHIBITION OF APOPTOSIS IN TUMOR CELLS THAT OVER-EXPRESSTHIOREDOXIN

The redox protein thioredoxin plays an important role in controllingcancer cell growth through regulation of DNA synthesis and transcriptionfactor activity. Thioredoxin is overexpressed by a number of humanprimary cancers and its expression is decreased duringdexamethasone-induced apoptosis of mouse WEH17.2 thymoma cells. Weexamined the ability of WEH17.2 cells stably transfected with humanthioredoxin cDNA showing increased levels of cytoplasmic thioredoxin toundergo apoptosis in vitro and in vivo. The cells were protected fromapoptosis induced by dexamethasone, staurosporine, etoposide, andthapsigargin, but not by N-acetyl-sphingosine. When inoculated intosevere combined immunodeficient mice, the trx-transfected cells formedtumors that showed increased growth compared to wild-type, as w ell asbcl-2-transfected. WEHI7.2 cells. The trx- and bcl-2-transfected celltumors both showed less spontaneous apoptosis than tumors formed by thewild-type cells. Unlike tumors formed by the wild-type andbcl-2-transfected WEH17.2 cells, frx-transfected cell tumors did notshow growth inhibition upon treatment with dexamethasone. This studysuggests that increased thioredoxin expression in human cancers mayresult in an increased tumor growth through inhibition of spontaneousapoptosis and a decrease in the sensitivity of the tumor to drug-inducedapoptosis.

Trx is a low molecular weight redox protein found in both prokaryoticand eukaryotic cells (Holmgren, A. J. Biol. Chem., 264:13963-13966,1989). The cysteine residues at the conserved Cys³²-Gly-Pro-Cys³⁵-Lysactive site of Trx undergo reversible oxidation-reduction catalyzed bythe NADPH-dependent selenium-containing flavoprotein Trx reductase(Luthman, M., et al., Biochemistry, 21:6628-6633, 1982). Human Trx is aprotein of M_(r) 11,500 with 27% of amino acid identity to Escherichiacoli but containing three additional Cys residues not found in bacterialTrx that give the human protein unique biological properties (Gasdaska,P. Y, et al., Biochem. Biophys. Ada., 1218:292-296, 1994). Trx wasoriginally studied for its ability to act as a reducing cofactor forribonucleotide reductase, the first unique step in DNA synthesis(Laurent, T. C., et al., J. Biol. Chem., 239:3436-3444, 1964). Morerecently, Trx has been shown to exert redox control over a number oftranscription factors, including nuclear factor KB (Matthews J R., et.al., Nucleic Acids Res., 20:3821-3830, 1992), transcription factor IIIC(Cromlish, J. A, et al., J. Biol. Chem., 264:18100-18109, 1989), BZLF1(Bannister, A. J, et al., Oncogene, 6:1243-1250, 1991), and theglucocorticoid receptor (Grippo, J. F, et al., J. Biol Chem.,258:13658-13664, 1983), and indirectly, through nuclear redox factorRef-1/HAPE, Trx can regulate AP-1 (Fos/Jun heterodimer; Abate, C, etal., Science (Washington, D.C.), 249:1157-1161, 1990).

Trx is also a growth factor with a unique mechanism of action. Human Trxstimulates the proliferation of both normal fibroblasts and a widevariety of human solid and leukemic cancer cell lines (Powis, G, et al.,On col. Res., 6:539-544, 1994; Oblong, J. E, et al., J. Biol. Chem.,269:11714-11720, 1994). Redox activity is essential for growthstimulation by Trx, and mutant redox-inactive forms of Trx lacking theactive site Cys³² and Cys³⁵ residues are devoid of growth stimulatingactivity (Oblong. J. E, et al., J. Biol Chem. 269:1.1714-11720, 1994).Studies with ¹²⁵I-labeled Trx have revealed no high-affinity bindingsites that might suggest receptors for Trx on the surface of cancercells (Gasdaska, J. R, et al., Cell Growth Differ., 6:1643-1650, 1995).Trx appears to stimulate cell proliferation by increasing thesensitivity of the cells to growth factors secreted by the cellsthemselves (Gasdaska, J. R. et al., Cell Growth Differ., 6:1643-1650,1995).

It has been found that Trx mRNA is elevated compared to paired normaltissue in almost half of the human primary lung and colon tumors weexamined (Gasdaska, P. Y, et al., Biochem. Biophys. Ada, 1218:292-296,1994; Berggren, M, et al., Anticancer Res., 16:3459-3466, 1996). Otherstudies have found increased Trx in human neoplastic cervical squamousepithelium cells and hepatocellular carcinoma (Fujii, S, et al., Cancer(Phila.), 68:1583-1591, 1991; Kawahara, N, et al., Cancer Res.,56:5330-5333, 1996). It has recently been shown that human breast cancercells transfected with a dominant negative, redox-inactive mutant Trxshow reduced anchorage-independent growth in vitro and an almostcomplete inhibition of tumor formation in vivo (Gallegos, A, et al.,Cancer Res. 56:5765-5770, 1996). Thus, Trx overexpression may be afactor in the growth of some human cancers.

It has previously been reported that Trx gene expression is decreasedduring dexamethasone-induced apoptosis of mouse thymoma-derived WEHI7.2cells (Briehl M, M, et al., Cell Death Differ., 2:41-46, 1995). Tofurther study the effects of Trx on apoptosis, in this study we stablytransfected WEHI7.2 cells with human Trx cDNA and examined the effectson both spontaneous and drug-induced apoptosis in vitro and with thecells growing in Scid mice.

a. Materials and Methods

(i) Cells

Human wild-type Trx cDNA was prepared as described previously, clonedinto the NotI site of the pDC304neo mammalian transfection vector(Gallegos, A, et al., Cancer Res., 56:5765-5770, 1996) and transfectedby electroporation into mouse WEH17.2 thymoma-derived cells (Harris, A.W, et al., J. Immunol, 110:431-438, 1973). Transfected cells weremaintained at culture densities up to 10 cells/ml in DMEM containing 10%fetal bovine serum supplemented with 800 μg/ml G418 sulfate. All studieswere conducted on clonal lines between passages 3 and 20. Stablytransfected bcl-2 WEH17.2 cells (W.Hbl2 cells) were obtained from Dr.Roger Miesfeld (University of Arizona, Tucson, Ariz.; Lam, M, et al.,Proc. Natl. Acad. Sci. USA, 91:6569-6573, 1994). Drugs were added at aculture density of 1×10⁵ to 2×10⁵ cells/ml. Stock solutions (10 mm) ofdexamethasone were prepared in ethanol, whereas staurosporine,etoposide, thapsigargin, and N-acetyl-sphingosine were prepared in DMSO.Further dilutions were made using culture medium.

(ii) mRNA Expression

Northern blot hybridization analysis was performed as describedpreviously using a full-length [o-³²P]dCTP-labeled human Trx cDNA probe(Gasdaska P Y, et al., Biochem. Biophys. Ada., 1218:292-296, 1994).Blots were quantified using a Molecular Dynamics PhosphorImager.

(iii) Glucocorticoid Receptors

The level of functional glucocorticoid receptors was assessed using atransient cotransfection of cells with a glucocorticoid responseelement/chloramphenicol acetyltransferase (“CAT”) reporter plasmid(pmmCAT; Rundlett, S. E, et al., Exp. Cell Res., 203:21.4-221, 1992) andP-galactosidase. After a 22-h recovery period, the cells were treatedwith 1 μM desamethasone, and CAT protein was measured after anadditional 24 hr. using a CAT ELISA (Boehringer Mannheim, Indianapolis,Ind.). An aliquot of the transfected cells was stained forp-galactosidase activity and CAT activity normalized for transfectionefficiency.

(iv) Apoptosis

Apoptosis was measured by an ELISA for histone-associated DNA fragments(Leist, M, et al., Biochemica., 11:20-22, 1994), by morphology and byflow cytometry (Philpott, N. H, et al., Blood, 6:2244-2251, 1996). Thecriteria used for the morphological identification of apoptotic cellsincluded condensation and margination of the chromatin with theformation of crescents, cell shrinkage, increased staining, nuclearfragmentation, cytoplasmic vacuolization, and apoptotic body formation.Cells were incubated with 20 μg/ml 7-amino actinomycin D for 30 min at4° C. before being analyzed by flow cytometry.

(v) Immunofluorescence Staining

Cells were centrifuged onto 0.17-mm-thick quartz coverslips, air driedfor 10 min. fixed with 4% methanol-free formaldehyde for 20 min at roomtemperature, washed for 15 min in PBS, pH 7.2, and permeabilized with100% methanol at −20° C. for 6 min. The coverslips were then stored at−20° C. until immunostaining, when they were allowed to come to roomtemperature and blocked with 1% BSA in PBS. This was followed by a 1:10dilution of goat serum in PBS, before reacting for 1 h with a 1:100dilution of immunoaffinity purified rabbit antihumanTrx polyclonalantibody (Berggren, M, et al., Anticancer Res., 1.6:3459-3466, 1996).After being washed with PBS, the coverslips were exposed to a 1:100dilution of goat antirabbit biotinylated IgG for h, washed with PBS,exposed to a 1:50 dilution of fluorescein streptavidin fluorochrome, andagain washed with PBS. Cells were examined using a Leica TCS-4D laserscanning confocal microscope with an excitation wavelength of 488 nm.For subcellular localization studies of Trx, Cy5(indodicarbocyanine)streptavidin was used as the fluorochrome, followed by digestion for 1 hat room temperature with 100 μg/ml RNase A and DNA stained with 25 nMYOYO-1 iodide for 10 min. Cells were then examined by laser scanningconfocal microscopy at excitations of 488 nm (YOYO-1) and 647 nm (Cy5).Relative fluorescence intensities of groups of 20 cells were measured atthe same laser power, photomultiplier tube voltage, and line averagingsetting as gray level intensities using SigmaScan software (JandelScientific, Corte Madera, Calif.), Because the transfected cellsexhibited an uneven distribution of fluorescent staining, a template ofa regular array of dots was placed over the image, and multiple (up to90) nuclear and cytoplasmic measurements were made.

(vi) In Vivo Tumor Growth

Tumor formation by wild-type and transfected WEH17.2 cells was studiedby injecting 2×10⁷ cells in 0.1 ml of matrigel s.c. into the flanks ofgroups of 20 female Scid mice. Tumor volume was measured with calipers,and mice were euthanized when the tumor volume exceeded 2 cm³. Nine daysafter tumor cell injection, 10 mice from each group were injected i.p.with 1 mg/kg/day dexamethasone in 10% ethanol in 0.9% NaCl. Control micewere injected with vehicle alone. On day 14, three mice from each groupwere euthanized with CO₂ and the tumors were excised and immediatelyfixed in glutaraldehyde.

(vii) Preparation of Tissue for Bright-Field Examination

The glutaraldehyde-fixed tissue was postfixed in osmium tetroxide,dehydrated in a graded series of alcohols, and embedded in epoxy resin.One-μm-thick sections were prepared and stained with toluidine blue forbright-field examination.

b. Results

WEHI7.2 cells were stably transfected with human trx cDNA in thepDC304neo mammalian transfection vector. I examined multiple clones andfound the maximal increase in Trc mRNA compared to endogenous levels ofmouse Trx mRNA, was 1.8-fold for clones Trx5 and Trx6 (FIG. 20A). Asdetermined by immunofluorescent staining and confocal microscopy, thetrx-transfected cells showed increased levels of Trx (FIG. 20B). Therelative fluorescence intensity of wild-type WEHI7.2 cells (±SE; n=20)was 1.00±0.05; of Trx5 cells, 2.15±0.14 (P<0.001 compared to wild type);and of Trx6 cells, 1.87±0.11 (P<0.001 compared to wild type). Trx-likefluorescent staining was observed in the nucleus as well as thecytoplasm of the cells (FIG. 20C). In the wild-type cells, 60.1±5.1% ofthe fluorescent staining was in the nucleus, in the Trx5 cells it was59.8±2.5%, and in the Trx6 cells it was 36.1±1.8%.

Compared to both wild-type or vector-alone-transfected cells, thetrx-transfected WEH17.2 cells were resistant to apoptosis induced by 1uM dexamethasone as measured by histone-associated DNA fragmentation(FIG. 21A) or by flow cytometry (FIG. 21B). Histological examination ofthe WEHI7.2 cells revealed a classic apoptotic morphology in response todexamethasone. However, only a small fraction of the cells undergoapoptosis at any one time, and they rapidly progress to fragmentedcells. For this reason, results are expressed as relative apoptosisrather than percentage of apoptotic cells. Glucocorticoid receptoractivity measured using a glucocorticoid receptor/CAT reporter plasmidwas not decreased in the toc-transfected cells (Results of three studiesnot shown). I also studied the effect of trx transfection on otheragents known to induce apoptosis (Table 5). Compared tovector-alone-transfected cells, fr-jc-transfected cells were resistantto apoptosis induced by staurosporine, a general kinase inhibitor(Kondo, Y, et al., Cancer Res., 55:2021-2023, 1995); by a cell-permeantsphingosine analogue, JV-acetyl sphingosine (Pushkareva, M, et al.,Immunol. Today, 16:294-297, 1995); by thapsigargin, which blocks theuptake of intracellular Ca²⁺ resulting in an increase in intracellularfree Ca²⁺ concentration (Lam, M, et al., Proc. Natl. Acad. Sci. USA,91:6569-6573, 1994); and by etoposide, a topoisomerase II inhibitor(Onishi, Y, et al. Biochem. Biophys. Ada., 1175:147-154, 1993). WEHI7.2cells transfected with the bcl-2 antiapoptotic proto-oncogene (W.HB 12cells) showed a similar pattern of protection against apoptosis inducedby the various agents as did the toc-transfected cells (Table 5).

TABLE 5 Effect of Trx and Bcl-2 Transfection On Apoptosis Induced byDifferent Agents Relative Apoptosis Neo Trx5^(a) Trx6^(a) W.Hb 12^(a)Dexamethasone 19.2 ± 0.6 4.4 ± 0.7 7.7 ± 1.2 0.7 ± 0.0 Staurosporine58.3 ± 9.8 5.3 ± 0.0 9.9 ± 1.1 4.8 ± 0.7 N-Acetyl-sphingosine 58.8 ± 7.111.1 ± 9.8  22.6 ± 4.5  5.5 ± 1.3 Etoposide 162.5 ± 12.1 6.0 ± 0.6 20.9± 4.1  4.5 ± 0.7 Thapsigargin  4.3 ± 0.9 2.3 ± 1.8 1.8 ± 1.2  0.9 ± 0.61pDC304neo vector-alone-transfected WEH17.2 cells (Neo),bcl-2-transfected WEH17.2 cells (W.Hbl2), and Trx5 and Trx-6trx-transfected WEH17.2 cells were treated with 1 μM dexamethasone for48 h, 100 nM staurosporine for 21 hr., 100 μM N-acetyl-sphingosine for24 hr., 1 μM etoposide for 15 hr., or 50 nM thapsigargin for 24 hr. Thetimes were determined to be optimum for detecting apoptosis with eachagent. Apoptosis was measured by flow cytometry as in FIG. 2 IB.Relative apoptosis is expressed as the ratio of the sum of regions R2and R3 (early and late apoptotic cells, respectively) divided by regionRI (live nonapoptotic cells) normalized to the ratio for vehicle treatedvector-alone transfected cells. Values are the mean of 3 determinations±SE. Statistical analysis was by linear regression with indicator valuesfor drugs and cells using the Stata statistical package (Stat Corp.,College Station, TX). ^(a)P < 0.05 compared to vector-alone transfectedcontrol cells.

When inoculated into Scid mice, the trx-transfected WEH17.2 cells formedtumors that grew more rapidly than tumors formed by either wild-type orbcl-2-transfected WEH17.2 cells (FIG. 22A). Upon histologicalexamination, tumors formed by the wild-type cells showed fields ofapoptotic cells adjacent to fields of viable cells, as well as apoptoticcells admixed with viable-appearing cells (FIG. 22B). The cellsundergoing apoptosis exhibited the classic appearance of condensed andmarginated chromatin, some in the form of crescents, and a densecytoplasm accompanied by vacuolization. The trx-transfected WEHI7.2 celltumors showed minimal numbers of cells undergoing apoptosis scatteredthroughout the tumor mass. Tumors formed by bcl-2-transfected WEHI7.2cells also showed very few cells undergoing apoptosis (not shown). Areasof necrosis were seen in wild-type, trx-transfected, andbcl-2-transfected cell tumors, usually adjacent to fields ofviable-appearing tumor cells or, in the case of the wild-type cells,adjacent to areas that show extensive apoptosis or next toviable-appearing cells. Treatment of the mice with dexamethasonestarting at day 9 had no effect on the growth of the trx-transfectedcell tumors but markedly inhibited the growth of the wild-type tumorsand the bcl-2-transfected cell tumors (FIG. 22A). Histologicalexamination revealed no evidence of increased apoptosis caused bydexamethasone treatment of wild-type, frx-transfected, orbcl-2-transfected cell tumors.

c. Discussion

WEH17.2 cells stably transfected with human trx showed a maximalincrease of 1.8-fold in Trx mRNA compared to endogenous levels of mouseTrx mRNA. This relatively low level of overexpression is similar to theexperience with trx transfection of mouse NIH 3T3 cells and human MCF-7breast cancer cells (Gallegos, A, et al., Cancer Res., 56:5765-5770,1996), suggesting that higher levels of unregulated trx expression maybe toxic to cells. As determined by immunofluorescent staining andconfocal microscopy, the frx-transfected cells showed approximately2-fold increased levels of Trx. The finding that Trx is present in thecytoplasm and the nucleus of cells confirms an earlierimmunohistochemical study using conventional light microscopy ofcervical tumor cells that reported cytoplasmic, nuclear, or cytoplasmicand nuclear localization of Trx (Fujii, S, et al., Cancer (Phila),68:1583-1591, 1991). This is an important observation because Trx may beable to directly reduce redox-regulated nuclear transcription factors,such as AP-1 (Fox/Jun heterodimer; Abate, C, et al., Science(Washington, D.C.), 249:1157-1161, 1990). If Trx can enter the nucleus,it may not need other nuclear redox factors such as Ref-1/HAP1, as hasbeen suggested (Abate, C, et al., Science (Washington, D.C.), 249:157-1161, 1990).

The trx-transfected cells were resistant to apoptosis induced bydexamethasone. Trx has been reported to be necessary for assembly of theglucocorticoid receptor (Grippo, J. F, et al., J. Biol. Chem.,258:13658-13664, 1983). However, glucocorticoid receptor activity wasnot decreased in the transfected cells, suggesting that the effects ofTrx on apoptosis appear to lie downstream of the glucocorticoidreceptor. The trx-transfected cells also showed resistance to apoptosisinduced by staurosporine etoposide, JV-acetyl sphingosine, andthapsigargin. Exogenously added human trx has been reported to inhibitapoptosis induced by tumor necrosis factor a in U937 human lymphomacells (Matsuda, M, et al., J. Immunol., 147:3837-3841, 1991). However,it has been found that exogenously added human Trx did not protectWEHI7.2 cells against apoptosis induced by dexamethasone (Baker, A R etal., Cell Death Differ, 3:207-213, 1996). Tumor necrosis factor a anddexamethasone are thought to trigger apoptosis by different signalingpathways. It may also be that exogenous Trx is not taken up by WEH17.2cells. We have found that other tumor cells take up Trx poorly, if atall (Gasdaska, J. R. et al., Cell Growth Differ., 6:1643-1650, 1995).Clearly, an increase in intracellular Trx achieved by transfection oftrx in the present study is associated with resistance of the WEH17.2cells to apoptosis induced by dexamethasone and other agents.

The pattern of resistance to drug-induced apoptosis caused by a trxtransfection is similar to that produced by transfection with the humanproto-oncogene bcl-2. Bcl-2 is believed to exert its inhibitory effectsupstream of the activation of the cysteine aspartate proteases cascade(caspase) responsible for the final stages of apoptosis (Shimizu, S, etal., Oncogene. 12:2251-2257, 1996). The protective effects of Bcl-2against apoptosis have been suggested to involve an antioxidantmechanism (Hockenberry, D. M, et al. Cell, 75:241-251, 1993), althoughthis is disputed based on the ability of Bcl-2 to block apoptosis causedby agents that are thought not to act by an oxidant mechanism (Jacobsen,M. D, et al., EMBO J., 13:1899-1910, 1994) or caused by hypoxia(Jacobsen, M. D, et al., Nature (Lond.), 374:814-816, 1995). Theantioxidants N-acetyl-cysteine, pyrrolidine dithio-carbamate. Trolox. (awater-soluble vitamin E analogue), and butylated hydroxytoluene protectrat thymocytes against drug-induced apoptosis (Wolfe, J. T, et al., FEBSLett., 352:58-62, 1994; Salgo, M. G, et al., Arch. Biochem. Biophys.,333:482-488, 1996). We have previously reported that Trolox, catalase,and superoxide disimutase protect murine WEH17.2 cells againstdexamethasone-induced apoptosis (Baker, A. F, et al., Cell DeathDiffer., 3:207-213, 1996). It is intriguing, therefore, that trx, a genethat codes for a known redox-active protein, also inhibits apoptosis.The mechanism by which Trx inhibits apoptosis remains to be established,but its pattern of antiapoptotic activity similar to Bcl-2 suggests thatit also may act upstream of the cysteine proteases.

WEH17.2 cells transfected with trx formed tumors in Scid mice that grewconsiderably faster than tumors formed by the wild-type parental cellsor by bcl-2-transfected cells. This may be due, in part, to a decreasedrate of spontaneous apoptosis that occurred in the frx-transfected celltumors. High levels of Bcl-2 have been found in a wide variety of humancancers (Reed, J. C, et al., J Cell. Biochem., 60:23-32, 1996). Althoughtransfection with bcl-2 is known to confer resistance to apoptosisinduced by anticancer drugs and radiation, the effects of bcl-2 on tumorgrowth are less clear. Transfection with bcl-2 gives a survivaladvantage to cells in culture (Maeyama, Y. Kurume Med. J., 42:291-297,1995). Transgenic mice overexpressing Bcl-2 under transcriptionalregulation of the immunoglobulin heavy chain enhancer develop benignlymphoma that eventually progresses to high-grade malignant disease(McDonnell, T. J, et al., Nature (Lond.), 349:254-256, 1991). Thissuggests that bcl-2 also provides a survival advantage to cells in vivabut that an additional change, most frequently rearrangement of myc(McDonnell, T. J., et al., Nature (Lond.), 349:254-256, 1991), isnecessary for tumor growth. Our studies using WEHI17.2 thymoma cellsshow that bcl-2-transfected cells formed tumors that grew faster thantumors formed by wild-type WEH17.2 cells. This may be due to a reductionin the rate of spontaneous apoptosis observed in the bcl-2 transfectedcell tumors compared to the wild-type tumors. It was not possible todistinguish a difference in the rates of spontaneous apoptosis betweenthe trx and bcl-2-transfected cell tumors. Paradoxically, thebcl-2-transfected cell tumors still showed growth inhibition byhigh-dose dexamethasone treatment, as did wild-type cell tumors. Therewas no evidence for increased apoptosis caused by dexamethasonetreatment of wild-type, rrx-transfected, or bcl-2-transfected celltumors, so the possibility remains that in vivo dexamethasone does notinhibit tumor growth in vivo by a mechanism that involves increasing therate of apoptosis.

The results of this study and our previous work (Gallegos, A, et al.,Cancer Res., 56:5765-5770, 1996) suggest that the Trx system offers anovel target for agents to promote apoptosis and inhibit tumor growth,as well as to reverse the drug resistance of some cancers. It isinteresting, therefore, that some 2-imidazolyl disulfide inhibitors ofTrx (Kuperus, M, et al., Proc. Am. Assoc. Cancer Res., 36:426, 1995)have been shown to induce apoptosis in cancer cells and, in animalstudies, by intraperitoneal and oral administration, to have antitumoreffects. In particular, 1-methylpropyl-2-imidazolyl disulfide exhibitsdoes-dependent antitumor activity in mice. Oral administration of1-methylpropyl-2-imidazolyl disulfide in the diet of mice at up to 250ppm, reduces the number of tumors in the colon by 70%, and causes asignificant reduction in the size of the remaining tumors, Injection ofmice with 1-methylpropyl-2-imidazolyl disulfide at 5 mg/kg, 10 mg/kg,and 15 mg/kg reduces the volume of tumors significantly (Powis, G, etal., Anticancer Drugs, 7 (Suppl. 3); 121-126, 1996).

In summary, it has been shown that transfection with r, a gene found tobe overexpressed in a number of human cancers, can inhibit apoptosis ofcancer cells in culture included by a variety of agents. In animals, thetrx-transfected cancer cells show an increased growth, decreasedspontaneous apoptosis, and decreased sensitivity to apoptosis induced bydexamethasone. If similar effects occur in patient tumors, then trxcould be a new human proto-oncogene.

8. THIOREDOXIN, A PUTATIVE ONCOGENE PRODUCT, IS OVER-EXPRESSED INGASTRIC CARCINOMA AND ASSOCIATED WITH INCREASED AND DECREASED APOPTOSIS

Human thioredoxin is a putative oncogene that may confer both a growthand survival advantage to tumor cells. Over-expressed thioredoxin mRNAhas been found in both primary human lung and colorectal cancers. Todetermine the intratumor distribution and amount of thioredoxin proteinin human primary tumors and to determine if its overexpression isrelated to proliferation or apoptosis, I studied primary human gastriccarcinoma samples. An immunohistochemical assay for thioredoxin inparaffin embedded blocks was developed. Ten patients were studied withprimary high risk gastric carcinoma. To relate thioredoxin proteinoverexpression to apoptosis I utilized a paraffin based in situ assay(Tunel) and to delineate proliferation we utilized the nuclearproliferation antigen detected by Ki67. In this survey I foundthioredoxin was localized to tumor cells and overexpressed compared tonormal gastric mucosa in 8 of 10 gastric carcinomas. The thioredoxin wasfound at high levels in 5 of the 8 overexpressing carcinomas. Theoverexpression of thioredoxin was typically found in both a nuclear andcytoplasmic location in the neoplastic cells. There was a significantpositive correlation (P=0.0061) with cancer cell proliferation measuredby Ki67. There was a significant negative correlation (P=0.0001) withapoptosis measured by the Tunel assay. Thus, human primary gastrictumors that are highly expressive of thioredoxin have both a higherproliferative rate and a lower rate of spontaneous apoptosis than tumorsthat do not express thioredoxin. Whether this thioredoxin-relatedcombined growth and survival advantage translates into poor clinicaloutcome remains to be determined.

Thioredoxins are low molecular weight redox proteins found in bothprokaryotic and eukaryotic cells (Holmgren, A. 1989. J. Biol. Chem.,264:13963-13966). The cysteine (Cys) residues at the conserved-Cys-Gly-Pro-Cys-Lys active site of thioredoxin undergo reversibleoxidation-reduction catalyzed by the NADPH-dependent selenium containingflavoprotein thioredoxin reductase (Luthman, M, et al., Biochem.,21:6628-6633, 1982). Human thioredoxin is an 11.5 kDa protein, with 27%amino acid identity to E. coli thioredoxin. It contains 3 additional Cysresidues not found in bacterial thioredoxin that give it uniquebiological properties (Gasdaska, P. Y, et al., Biochem. Biophys. Ada.,1218:292-296, 1994).

Thioredoxin was first studied for its ability to act as reducingcofactor for ribonucleotide reductase, the first unique step in DNAsynthesis (Laurent, T. C, et al., J. Biol. Chem., 239:3436-3444, 1964).More recently thioredoxin has been shown to exert redox control over anumber of transcription factors, including NF-KB (Matthews, J. R, etal., Nucl. Acids Res., 20:3821-3830, 1992). TFIHC (Cromlish, J. A, etal., J. Biol. Chem., 264:18100-18109, 1989), BZLF1 (Bannister, A. J, etal., Oncogene, 6:1243-1250, 1991), the glucocorticoid receptor (Grippo,J. F, et al., J. Biol. Chem., 258:13658-13664, 1983) and, indirectlythrough another redox factor Ref-1, AP-1 (Fos/Jun heterodimer)(Bannister, A. J, et al., Oncogene, 6:1243-1250, 1991). Thioredoxinmodulates the binding of the transcription factors to DNA and thus,regulates gene transcription.

Thioredoxin is also a growth factor with a unique mechanism of action.The predicted amino acid sequence of thioredoxin is identical to that ofa previously identified growth factor secreted by HTLV-1 transformedleukemic cell lines, called adult T-cell leukemia-derived factor (ADF)(Gasdaska, P. Y. et al., Biochem. Biophys. Ada., 1218:292-296, 1994).ADF stimulates growth of lymphoid cells (Wakasugi, N, et al., Proc Natl.Acad. Sci. USA, 87:8282-8286, 1990; Yodoi, J, et al., Adv. Cancer Res.,57:381-411, 1991). It has been shown that human recombinant thioredoxinstimulates the proliferation of normal fibroblasts and human solid tumorcancer cells even in the absence of serum (Powis, G, et al., Oncol Res.,6:539-544, 1994; Oblong, J. E, et al., J. Biol. Chem., 269:11714-11720,1994). It does this by increasing the “sensitivity of the cells togrowth factors secreted by the cells themselves (Gasdaska, J. R, et al.,Cell Growth Differ., 6:1643-1650, 1995). For example thioredoxin at nMconcentrations, as are found in human serum (Kitaoka, Y, et al.,Immunol. Lett., 41:155-161, 1994), increases the sensitivity of humanbreast cancer cells to interleukin-2 (1-2) and basic fibroblast growthfactor (bFGF) by 1000 and 100 fold, respectively (unpublishedobservations). The term “voitocrine”, from the Greek “to help”, has beencoined to describe this growth stimulating activity of thioredoxin(Gasdaska, J. R, et al., Cell Growth Differ., 6:1643-1650, 1995). Mutantredox-inactive forms of thioredoxin lacking the active site cysteineresidues and E. coli thioredoxin are devoid of growth stimulatingactivity (Oblong, J. E, et al., J. Biol. Chem., 269:11714-11720, 1994).Human thioredoxin is known to be secreted from cells by a leaderlesssecretory pathway (Rubartelli, A, et al., J. Biol. Chem.,267:24161-24164, 1992) so that it could be acting extracellularly tostimulate cancer cell growth.

Our work has shown that thioredoxin is important for the growth, deathand transformed phenotype of some human cancers. Stable transfection ofnormal fibroblasts with human thioredoxin cDNA (trx) increases theirgrowth rate and transfection of human MCF-7 breast cancer cells with trxincreases their colony formation in soft agarose (Gallegos, A., et al.,Cancer Res., 56:5765-5770, 1996). Transfection of the MCF-7 cells with adominant negative redox inactive mutant trx causes inhibition of colonyformation and almost complete inhibition of tumor formation when thecells were inoculated into Scid mice. In recent studies 1 have shownthat stable transfection of mouse thymoma cells with human trx inhibitsapoptosis induced by a variety of agents including glucocorticoid,staurosporine, N-acetylsphingosine, thapsigargin and etoposide, which issimilar to the pattern of inhibition seen with the antiapoptoticoncogene bcl-2 in these cells (Baker, A, et al. Cancer Research,57:5162-5167, 1996). The trx transfected cells form tumors that wheninoculated in Scid mice grow more rapidly and show less spontaneousapoptosis than vector alone or bcl-2 transfected cells, and areresistant to growth inhibition by glucocorticoid (Baker, A, et al.,Cancer Research, 57:5162-5167, 1996). These results suggest that trxoffers a survival as well as a growth advantage to tumors in vivo,unlike bcl-2 which offers only a survival advantage and requires othergenetic changes for tumor growth (McDonnell, T J, et al., Nature,349:254-256, 1991).

It has previously been reported that almost half of human primary lungcancers examined overexpress thioredoxin mRNA compared to normal lungtissue from the same subject (Gasdaska, P. Y, et al., Biochem. Biophys.Acta., 1218:292-296, 1994). Recently it has been found that more thanhalf of human primary colorectal tumors have elevated levels ofthioredoxin mRNA, up to over 100 fold for one subject, compared tonormal colonic mucosa taken from within 5 cm of the tumor from the samesubject (Berggren, M, et al., Anticancer Res., 16:3459-3466, 1996). Inthese studies, however, thioredoxin mRNA was extracted from pieces oftumor and nothing is known of its intratumor distribution, or even ifthe increased thioredoxin mRNA leads to an increase in thioredoxinprotein. It remains to be determined if thioredoxin overexpression isrelated to proliferation or apoptosis in human primary tumors. These areclearly important questions that are now addressed in the presentstudies utilizing primary human gastric carcinoma samples.

The current study sought to develop an assay for thioredoxin in paraffinembedded blocks allowing survey of human tumors in archival tissuebanks. To this end the Southwest Oncology Group (SWOG) GastrointestinalBiology Laboratory made available a relevant archival paraffin blockbank of gastric carcinomas. Furthermore, to relate thioredoxin toapoptosis I also sought to refine a paraffin-based in situ assay ofapoptosis (Grogan, T. M, et al., Automation of Immunohistochemistry, InWeinstein, R. S. (ed); Advances in Pathology and Laboratory Medicine,vol. 6. St. Louis, Mosby, 1993, pp. 253-283; Grogan, T M, et al.Kinetic-mode, Automated Double-labeled Immunohistochemistry and In SituHybridization in Diagnostic Pathology, Advances in Pathology andLaboratory Medicine. 8:79-100, 1995). Finally, to relate thioredoxin toproliferation, I utilized the previously developed assay of the nuclearproliferation antigen detected by Ki67 (Miller, T, et al., Blood83:1460-1466, 1994).

a. Methods

(i) Patient Samples

Paraffin blocks from ten gastric carcinoma resections were studied.These pathology samples derived from ten patients on Southwest OncologyGroup (SWOG) protocol 9008 (also known as intergroup study #0116). Thisis a study of high risk gastric carcinoma comparing gastrectomy onlyversus gastrectomy plus adjuvant therapy. The patients ranging in agefrom 42 to 75; all had previously untreated, stage II and III B gastriccarcinoma. They had biopsy proven adenocarcinoma of the stomach whichhad a high risk for recurrences due to evidence of carcinoma extensionbeyond the muscularis propria and/or having lymph node involvement.Patients with Stage O, IA or any stage with MI were not eligible. As ofDecember 1996 this study has accrued 486 patients.

(ii) Immunohistochemistry

Five micron thick sections were deparaffinized and then subjected toantigen unmasking with one of two methods with heat plus citrate buffera pH 6.6 or microwave plus EDTA buffer at pH 8.0 as previously described(Grogan, T. M, et al., Automation of Immunohistochemistry, In Weinstein,R. S. (ed); Advances in Pathology and Laboratory Medicine, vol. 6. St.Louis, Mosby, 1993, pp. 253-283; Grogan. T. M, et al. Kinetic-mode,Automated Double-labeled Immunohistochemistry and In Situ Hybridizationin Diagnostic Pathology, Advances in Pathology and Laboratory Medicine,8:79-100, 1995). The best signal to noise ratio was established byjudging reactivity with cell lines known to be a high expressor ofthioredoxin (A549 human lung cancer) and a low expressor of thioredoxin(SK BR3 human breast cancer) (Berggren, M, et al., Anticancer Res.,16:3459-3466, 1996).

All tumor samples and control cell lines were stained using a standardimmunohistochemical method as previously described (Grogan, T. M, etal., Automation of immunohistochemistry, In Weinstein, R. S. (ed);Advances in Pathology and Laboratory Medicine, vol. 6. St. Louis, Mosby,1993, pp. 253-283; Grogan, T. M, et al. Kinetic-mode, AutomatedDouble-labeled Immunohistochemistry and In Situ Hybridization inDiagnostic Pathology, Advances in Pathology and Laboratory Medicine,8:79-100, 1995). To obviate biotin receptor reactivity, biotin-avidinblocking was performed first. Then the primary antibody (polyclonalrabbit anti-human thioredoxin) (Berggren, M, et al., Anticancer Res.,16:3459-3466, 1996) was utilized at a titer of 1/200 after titration ofcontrol cell lines. The best signal to noise ratio was found followingmicrowaving at pH 8.0 with EDTA buffer. Sections were treated withbiotinylated goat-anti-rabbit antibody and then with avidin-peroxidasecomplex, each for 30 minutes at 42° C. in an automated immunostainer(VMS ES, Ventana Medical Systems, Tucson, Ariz.) (Grogan, T. M, et al.,Automation of Immunohistochemistry, In Weinstein, R. S. (ed); Advancesin Pathology and Laboratory Medicine, vol. 6. St. Louis, Mosby, 1993,pp. 253-283; Grogan, T. M, et al. Kinetic-mode, Automated Double-labeledImmunohistochemistry and In Situ Hybridization in Diagnostic Pathology,Advances in Pathology and Laboratory Medicine, 8:79-100, 1995). Sectionswere counterstained with methyl green, dehydrated, rinsed in xylene andcoverslipped.

The degree of thioredoxin expression in tumor cells was judged at 400×magnification as 4+ (very intensely positive), 3+ (moderately intenselypositive), 2+ (moderate), 1+ (faint), or 0 (completely negative)throughout the sample. A single investigator (TG) was responsible forscoring all the samples.

Additional immunohistochemical assays employed antibody to proliferationantigens, Ki67 (Ventana, Tucson, Ariz.), also using the biotin-avidinlabelled method after avidin blocking (Miller, T, et al., Blood,83:1460-1466, 1994). The degree of Ki67 staining, again judged at 400×magnification, was classified as the percentage of nuclear positivetumor cells listed as: absent (0), >0-5% (+), 6-25% (++),26-50(+++), >51% (+++).

(iii) Apoptosis Assay

Apoptotic cells were detected utilizing the TUNEL assay (Gavrieli, Y, etal., J. Cell BioL, 119:493-501, 1992; Grasl-Kraupp, B. et al.,Hepatology, 21:1465-1468, 1995) adapted to an automated in situhybridization instrument (gen II, Ventana Medical Systems, Inc.). TheTUNEL assay utilizes recombinant terminal deoxynucleotidyl transferase(Tdr) (GIBCO BRL) for adding homopolymer tails to the 3′ ends of DNAwhich are more abundant in apoptotic cells (Gavrieli, Y. et al., J. CellBioL, 119:493-501, 1992; Grasl-Kraupp, B, et al. Hepatology,21:1465-1468, 1995). Biotin-16, 2′-deoxyuridine-5′ triphosphate (Biotin16-dUTP) (Boehringer-Mannheirn, Indianapolis, Ind.) was the label usedfor terminal transferase in this DNA 3′-end labelling reaction.Avidin-Horseradish Peroxidase and 3,3′-diaminobenzidine as chromogen(Gavrieli, Y, et al, J. Cell BioL, 119:493-501, 1992; Grasl-Kraupp, B,et al., Hepatology, 21:1465-1468, 1995).

The instrument utilized deparaffinized sections with subsequentdigestion with Protease I (Ventana Medical Systems, Tucson, Ariz.) for 8minutes VMS 1). Incubations were performed per Ventana Gen II protocolon the instrument with the final steps being as above using avidin-horseradish peroxidase and DAB detection method to visualize the apoptoticnuclei as an intense brown color (diaminobenzidine). As an enzymecontrol we utilized two sections from each tissue: one with Tdt enzymeand one without enzyme (negative control).

The TUNEL assay result was scored by the number of brown-apoptotic tumornuclei per high power field (400× objective). The values were: 0(absence of apoptotic cells), + (>0-2/hpf), ++ (2-4/hpf), +++(>4-8/hpf), ++++ (>8/hpf).

b. Statistical Analysis

Thioredoxin expression was correlated with Ki67 expression and withapoptosis measured by the TUNEL assay using Spearman's nonparametricrank correlation test.

c. Results

The optimum signal to noise ratio was found by using the followingantigen retrieval conditions: microwaving at pH 8.0 in EDTA as tested bya high thioredoxin expressor (A 549) and low thioredoxin expressing (SkBR3) cell line.

Immunohistochemical localization of thioredoxin (positive staining) wasfound in the tumor cells of 8 of 10 gastric carcinoma samples (Table 2,supra). Seven of these eight showed both nuclear and cytoplasmicstaining (FIG. 23). The two cases with no tumor thioredoxin showedpositive staining in the adjacent normal mucosa and are importantcontrols, suggesting these are true, not false-negative, tumors (FIG.24).

Among the eight thioredoxin positive gastric carcinomas there was arange of positivity from faint (+) to intense (++++) with five caseshaving high level thioredoxin (+++ to ++++) and three having low level(+ to ++) (Table 2, supra).

In all samples there was the adjacent normal mucosa where the strongeststaining was found in the gastric mucosal pits (++) while faint stainingwas found in the superficial mucosa (+). The localization differed basedon site with gastric pits showing both nuclear and cytoplasmic stainingwhile the middle mucosa had only cytoplasmic staining (FIG. 25).

Increased levels of thioredoxin levels positively correlated withincreased cell proliferation as measured by Ki67 expression (r=0.861,p=0.0061) and negatively correlated with apoptosis as measured by theTunel assay (r=0.949, p—0.00010 (see Table 2, and FIGS. 26 and 27).

d. Discussion

An important aspect enabling this study is the development of twomethodologic refinements: (Holmgren, A. 1989. J. Biol. Chem.,264:13963-13966) the use of heatbased antigen unmasking methods to allowoptimal, reliable measurement of thioredoxin by IHC in archival paraffinembedded tissues; and (Luthman, M., Holmgren, A. Biochem., 21:6628-6633)adaptation of the Tdt based TUNEL assay to an automated procedure on anautomated in situ machine. The heat-based antigen optimization of IHCentailed heating the paraffin section in 5 mM EDTA in 0.1 M TRIS, pH8.0. The specificity of the reaction was assured by the finding of highpositive signal in A549 human lung carcinoma, a known high levelthioredoxin expressor as determined by prior Western blotting (Berggren,M. et al, Anticancer Res., 16:3459-3466, 1996). SK BR3 breast carcinomacells likewise served as low level expressor control also established byprior Western blotting. The gastric tumor samples themselves also servedas positive and negative, same-slide controls. In particular, within theentrapped normal gastric mucosa gastric pits and mid-level mucosal cellsshowed thioredoxin signal while surface mucosal cells were negative.There was a clear difference in the subcellular localization ofthioredoxin in normal positive gastric cells, the lower level in thepits showed cytoplasmic and scattered nuclear staining, while the highermid-level graduation staining was typically lighter and restricted tothe cytoplasm. The significance of this differential distributions isnot known. Thioredoxin does not have a known nuclear localizationsequence (Gasdaska, P. Y, et al., Biochem. Biophys. Ada., 1218:292-296,1994). From our IHC studies it is clear that thioredoxin is specificallylocated within neoplastic gastric carcinoma cells and not in stromalcells or admixed B or T lymphocytes or macrophages. The tumor cellthioredoxin density typically exceeded that of the adjacent normalmucosa. The minimal background staining and strong signal to noise inall the samples, as illustrated in FIGS. 23-27, demonstrate therefinement of the thioredoxin paraffin assay we have developed.

In this survey of 10 primary human gastric carcinomas we have determinedthe extent of thioredoxin overexpression and determined its localizationand its relationship to proliferation and cell survival (apoptosis)status. We found thioredoxin is overexpressed, compared to normalgastric mucosa, in the malignant cells of 8 of 10 gastric carcinomas.The thioredoxin protein was found at high levels in five of the eightoverexpression carcinomas. The expression was typically found in both anuclear and cytoplasmic location in the neoplastic cells. There was asignificant positive correlation (p<0.01) between increased levels ofthioredoxin expression and cell proliferation measured by Ki67expression. There was also a significant negative correlation (p<0.0001)between increased levels of thioredoxin and apoptosis measured by theTUNEL assay. Thus, human primary gastric tumors highly expressive ofthioredoxin have both a higher proliferative rate and a lower rate ofspontaneous apoptosis than tumors with absent or low thioredoxin (FIG.26). This finding is consistent with our experimental observation thatthe stable transfection of mouse WEHI7.2 cells with human wild typethioredoxin leads to increased tumor growth rate in vivo associated witha decreased rate of spontaneous apoptosis (Baker, A, et al., CancerResearch, (in press) 1996). We have also found that transfection ofhuman MCF-7 breast cancer cells with a dominant-negative redox inactivemutant thioredoxin inhibits tumor growth in vivo (McDonnell, T. J, etal., Nature, 349:254-256, 1991). Thus, overexpression of thioredoxin ingastric carcinoma is associated with increased cell growth and cellsurvival giving the cells doubly immortalizing properties. Whether thiswill translate in patients into more aggressive tumor growth, as seen inanimals with thioredoxin transfected tumor cells, and a poor prognosisremains to be determined.

There have been 2 reports of the immunohistochemical distribution ofthioredoxin in human primary tumors, Fujii et al., Cancer,68:1583-11591, 1991 reported that, while the squamous and glandularcells of normal human cervix showed no thioredoxin IHC, the immediateand superficial layers of cervical squamous neoplastic tissue, as wellas invasive squamous cell carcinoma showed cytoplasmic and nuclearstaining for thioredoxin. A study by Kawahara, N, et al., Cancer Res.,56:5330-5333, 1996 has reported enhanced expression of thioredoxin inhuman hepatocellular carcinoma compared to adjacent non-cancerous liver,with both a nuclear and cytoplasmic localization of the staining. Thus,thioredoxin, overexpression appears to be a common phenomenon among adiversity of human neoplasms.

Future studies are required to confirm the relationship betweenthioredoxin overexpression, increased gastric cancer proliferation andincreased cell survival. The newly developed ability to simultaneouslyperform combined TUNEL and IHC assays on a single tissue section shouldallow more precise definition of the relationship of thioredoxin to cellproliferation or cell death, since the phenotype of individual apoptoticor proliferative cells may now be discerned by these double labelledassays.

Finally, correlative clinical studies are anticipated to relatethioredoxin expression, Ki67 and apoptosis index to pathogenic grade,response to chemotherapy, disease free survival or overall survival thusdefining the impact of thioredoxin on human carcinomas. Our patient dataon the SWOG 9008 (Intergroup 0116) study of high risk gastric carcinomaswhich is now ongoing and has accrued 486 gastric carcinoma patientswould seem to be the ideal patient cohort to study. Now that we havedeveloped paraffin-based assays for thioredoxin, Ki67 and Tdt apoptosisby the Tunel assay in a standardized optimized manner, the full clinicalstudy testing the clinical impact of thioredoxin is now feasible.

All of the various publications cited above are hereby incorporated byreference in their entireties.

1. A method of inhibiting tumor cell growth in a tumor cell thatover-expresses thioredoxin comprising contacting said tumor cell with acell growth inhibiting effective amount of an inhibitor of thioredoxinexpression.
 2. A method of reducing inhibition of apoptosis in tumorcells that over-express thioredoxin comprising contacting said tumorcells with an effective amount of an agent that inhibits thioredoxin. 3.A method of identifying an agent that inhibits tumor cell growth incells that over-express thioredoxin comprising measuring thioredoxinexpression in a first sample of said cells; contacting a second sampleof said cells with an agent to be tested; measuring expression ofthioredoxin in said second sample; comparing expression of thioredoxinin said first sample and said second sample; whereby a decrease inexpression of thioredoxin in said second sample is indicative of anagent that inhibits tumor cell growth.
 4. A method of identifying anagent that reduces inhibition of apoptosis in a tumor cell thatover-expresses thioredoxin comprising measuring thioredoxin expressionin a first sample of said cells; contacting a second sample of saidcells with an agent to be tested; measuring expression of thioredoxin insaid second sample; comparing expression of thioredoxin in said firstsample and said second sample; whereby a decrease in expression ofthioredoxin in said second sample is indicative of an agent that reducesinhibition of apoptosis.
 5. A method of identifying an agent thatreduces inhibition of apoptosis in a tumor cell growth.
 6. A method ofstimulating cell growth comprising introducing a nucleic acid encoding ahuman thioredoxin having Ser at amino acid reside 73 under conditionswhereby said nucleic acid is expressed.
 7. A composition comprising anagent that is useful in reducing or eliminating thioredoxin-associatedapoptosis inhibition and an acceptable carrier.
 8. A compositioncomprising an agent that is useful in inhibiting thioredoxin stimulatedcell growth and an acceptable carrier.